Methods for making and using molecular switches involving circular permutation

ABSTRACT

The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/588,114, filed Aug. 1, 2007, allowed, which is the U.S. nationalstage of PCT/US2005/002633, filed Jan. 28, 2005, which claims priorityof U.S. Provisional Patent Application Ser. Nos. 60/539,774, 60/557,152,60/607,684, and 60/628,997, filed Jan. 28, 2004, Mar. 26, 2004, Sep. 7,2004, and Nov. 18, 2004, respectively, the entire disclosures of whichare incorporated herein by reference in their entirety.

STATEMENT AS TO FEDERALLY SUPPORTED RESEARCH

The present invention was made with United States government supportunder grant number R01 GM066972-01A1 from the National Institutes ofHealth. Accordingly, the United States government has certain rights inthe invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 27, 2013, isnamed 62568DIV(71699)_SL.txt and is 192,029 bytes in size.

FIELD OF THE INVENTION

The invention relates to fusion molecules which function as molecularswitches and to methods for making and using the same. Moreparticularly, combinatorial methods involving circular permutation ofDNA are used.

BACKGROUND OF THE INVENTION

A hallmark of biological systems is the high degree of interactionsamong their constituent components. Cells can be described as complexcircuits consisting of a network of interacting molecules. Key componentof these networks are proteins that serve to couple cellular functions.A protein that couples functions can be described as a “molecularswitch.” In most general terms, a molecular switch recognizes aneffector (input) signal (e.g., ligand concentration, pH, covalentmodification) with resultant modification of its output signal (e.g.,enzymatic activity, ligand affinity, oligomeric state). Examples ofnatural molecular switches include allosteric enzymes that coupleconcentration of effector molecules with level of enzymatic activity,and ligand-dependent transcription factors that couple ligandconcentration to output level of gene expression. Molecular switches canbe “ON/OFF” in nature or can exhibit a graded response to a signal.

There is recognition that there is great potential to design fusionproteins that act as molecular switches to modulate or report onbiological functions for a variety of applications including biosensors(Siegel and Isacoff 1997; Baird, Zachariasiii et al. 1999; Doi andYanagawa 1999; de Lorimier, Smith et al. 2002; Fehr, Frommer et al.2002) modulators of gene transcription and cell signaling pathways(Rivera 1998; Guo, Zhou et al. 2000; Picard 2000), and novelbiomaterials (Stayton, Shimoboji et al. 1995). Despite its greatpotential, however, molecular switch technology has not been extensivelyexploited, in part due to technical challenges in engineering effectivemolecular switches. In general, existing approaches to creating proteinmolecular switches include: control of oligomerization or proximityusing chemical inducers of dimerization (CID); chemical rescue; fusionof the target protein to a steroid binding domain (SBD); coupling ofproteins to nonbiological materials such as ‘smart’ polymers (Stayton,Shimoboji et al. 1995; Ding, Fong et al. 2001; Kyriakides, Cheung et al.2002) or metal nanocrystals (Hamad-Schifferli, Schwartz et al. 2002);and domain insertion.

The approach of control using a chemical inducer of dimerization (CID)utilizes a synthetic ligand as the CID that controls the oligomeric orproximity of two proteins (Rivera 1998). CIDs are small molecules thathave two binding surfaces that facilitate the dimerization of domainsfused to target proteins. This approach was first developed using theimmunosupressant FK506 to facilitate dimerization of target proteinsfused to the FK506-binding protein, FKBP12 (Spencer, Wandless et al.1993). Several variations on this system have since appeared as well asa system using the antibiotic coumermycin to dimerize proteins fused toB subunit of bacterial DNA gyrase (GyrB) (Farrar, Olson et al. 2000).CIDs have been used to initiate signaling pathways by dimerizingreceptors on the cell surface, to translocate cytosolic proteins to theplasma membrane, to import and export proteins from the nucleus, toinduce apoptosis and to regulate gene transcription (Bishop, Buzko etal. 2000; Farrar, Olson et al. 2000). However, CIDs have only beenapplied to those functions that require changes in the oligomeric stateor proximity of the two proteins. As described in the literaturehowever, this approach cannot be readily applied to a single protein.

Chemical rescue has recently been applied as a strategy for control, inthe case of dimerization (Guo, Zhou et al. 2000). Chemical rescue aimsto restore activity to a mutant, catalytically defective enzyme by theintroduction of a small molecule that has the requisite properties ofthe mutated residues. Since first described for subtilisin (Carter andWells 1987), chemical rescue has been demonstrated for a number ofdifferent mutated protein-small molecule pairs (Williams, Wang et al.2000). The vast majority of these rescues required >5 mM concentrationsto show detectable rescue, and the maximum fold improvement in activityof the mutant was generally less than 100-fold and required >100 mMconcentrations of the rescuing molecule.

For the strategy of fusion to a steroid binding domain, the protein tobe controlled is fused end-to-end to a SBD (Picard 2000). In the absenceof the steroid that binds to the SBD, it is believed that a Hsp90-SBDcomplex sterically interferes with the activity of the protein fused tothe SBD. The disassembly of the complex upon steroid binding restoresactivity to the protein. This strategy has been successfully appliedprincipally to transcription factors and kinases (Picard 2000).Artificial transcription factors (such as GeneSwitch™) have beendeveloped using this strategy and have promise for tissue-specific geneexpression in transgenic animals and human gene therapy (Burcin, B W etal. 1998; Burcin, Schiedner et al. 1999).

For approaches involving coupling to non-biological materials, theprotein to be controlled is coupled to a non-biological material thatresponds to an external signal and thereby affects the protein coupledto it. ‘Smart’ polymers that change their conformation upon a change inpH or temperature have been conjugated to proteins near ligand bindingsites, to create switches that sterically block access to the bindingsite at, for example, higher temperatures, but not at lower temperatures(Stayton, Shimoboji et al. 1995; Ding, Fong et al. 2001). Inductivecoupling of a magnetic field to metal nanocrystals attached tobiomolecules resulting in an increase in local temperature therebyinducing denaturation, has so far only been applied to DNA(Hamad-Schifferli, Schwartz et al. 2002).

Relatively few studies have attempted to create a molecular switch usingthe approach of insertional fusion, in which one gene is inserted intoanother gene. Insertions result in a continuous domain being split intoa discontinuous domain. The first example of successful insertion of oneprotein into another was of alkaline phosphatase (AP) into the E. coliouter membrane protein MalF, constructed as a tool for studying membranetopology (Ehrmann, Boyd et al. 1990). High levels of alkalinephosphatase activity were obtained in the fusions despite the fact thatalkaline phosphatase requires dimerization for activity. Other examplesof proteins that have been inserted into other proteins include greenfluorescent protein GFP) (Siegel and Isacoff 1997; Biondi, Baehler etal. 1998; Kratz, Bottcher et al. 1999; Siegel and Isacoff 2000), TEM1β-lactamase (Betton, Jacob et al. 1997; Doi and Yanagawa 1999; Collinet,Herve et al. 2000), thioredoxin (Lu, Murray et al. 1995), dihydrofolatereductase (Collinet, Herve et al. 2000), FKBP12 (Tucker and Fields2001), estrogen receptor-α(Tucker and Fields 2001) and β-xylanase (AÿGötz et al. 1998).

In studies of insertions into GFP, molecular sensors were created byinserting β-lactamase into GFP by random mutagenesis, to create aprotein whose fluorescence increased 60% upon binding of the β-lactamaseinhibitory protein. Insertions of calmodulin (a Ca²⁺ binding protein)into GFP resulted in a fusion whose fluorescence changed up to 40% uponincreases in Ca²⁺ concentration (Baird, Zacharias et al. 1999). In arelated strategy, the gene for a circularly permuted GFP was sandwichedbetween the gene for calmodulin and its target peptide M13 to create aseries of sensors whose fluorescence intensity increased, decreased orshowed an excitation wavelength change upon binding Ca2⁺ (Nagai, Sawanoet al. 2001).

With the exception of the domain insertion strategy, all of theabove-described approaches to engineering a molecular switch are limitedin the sorts of signals that can be employed or the types of proteinsthat can be controlled. CIDs have only been applied to those functionsthat require changes in the oligomeric state or proximity of the twoproteins and thus cannot be used to control a single protein. Thechemical rescue approach is limited by the inability to apply the methodto any desired signal and by the lack of sensitivity (highconcentrations of the signal are required for a small change inactivity). The SBD strategy appears to be limited as a general methodfor controlling any protein due to the apparent requirement forend-to-end fusion.

The domain insertion strategy is a promising and generally applicableapproach to engineering a molecular switch. However existing domaininsertion strategies are limited by the number of possible insertionalfusions between the two domains. Generally, methods for generatingmolecular switches have not provided a systematic way to generate verylarge numbers of fusions of different geometries that would be ideal forgenerating and optimizing functional coupling of protein domains inmolecular switches.

SUMMARY OF THE INVENTION

The invention provides improved molecular switches, for example withswitching activity greater than previously demonstrated, or with alteredligand recognition and binding, and methods of making these moleculesinvolving circular permutationof nucleic acid or amino acid sequences.Molecular switches have been created by recombining nonhomologous genesin vitro and subjecting the genes to evolutionary pressure usingcombinatorial techniques. The approach may be envisioned as “rolling”two proteins across each other's surfaces and fusing them at pointswhere their surfaces meet. The approach allows for recombination andtesting of maximal numbers of geometric configurations between the twodomains. Libraries comprising vast numbers of such fused molecules areprovided from which molecular switches with optimal characteristics canbe isolated.

Preferred switches are fusion molecules comprising an insertion sequenceand an acceptor sequence for receiving the insertion sequence, whereinthe state of the insertion sequence is coupled to the state of theacceptor sequence. For example, the activity of the insertion sequencecan be coupled to the activity/state of the acceptor sequence.

The “state” of a molecule can comprise its ability or latent ability toemit or absorb light, its ability or latent ability to changeconformation, its ability or latent ability to bind to a ligand, tocatalyze a substrate, transfer electrons, and the like. Preferably,molecular switches according to the invention are multistable, i.e.,able to switch between at least two states. In one aspect, the fusionmolecule is bistable, i.e., a state is either “ON” or “OFF,” forexample, able to emit light or not, able to bind or not, able tocatalyze or not, able to transfer electrons or not, and so forth. Inanother aspect, the fusion molecule is able to switch between more thantwo states. For example, in response to a particular threshold stateexhibited by an insertion sequence or acceptor sequence, the respectiveother sequence of the fusion may exhibit a range of states (e.g., arange of binding activity, a range of enzyme catalysis, etc.). Thus,rather than switching from “ON” or “OFF,” the fusion molecule canexhibit a graded response to a stimulus. More generally, a molecularswitch is one which generates a measurable change in state in responseto a signal.

Accordingly, and in one aspect, the invention provides a method forassembling a fusion molecule, comprising: generating an insertionsequence by circular permutation; and inserting the insertion sequenceinto an acceptor sequence.

In one variation of the method, the insertion sequence is inserted at aselected site in the acceptor sequence. In another variation, theinsertion sequence is inserted at a random site in the acceptorsequence.

Another aspect of the invention is a method for assembling a modulatablefusion molecule, comprising: generating an insertion sequence bycircular permutation; inserting the insertion sequence into an acceptorsequence, wherein the insertion sequence and the acceptor sequence eachcomprise a state; and selecting a fusion molecule, wherein the state ofthe insertion sequence and the state of the acceptor sequence arecoupled. As in the above method, variations are provided wherein theinsertion sequence is inserted at a selected site in the acceptorsequence or at a random site in the acceptor sequence.

In some embodiments of the method, the state of the insertion sequencegenerated by circular permutation is modulated. The state of theinsertion sequence can be modulated in response to a change in the stateof the acceptor sequence, or modulated in response to a change in thestate of the insertion sequence. The fusion molecule can furthercomprise a new state.

In yet another aspect is provided a method for assembling a multistablefusion molecule which can switch between at least an active state and aless active state, comprising: generating an insertion sequence bycircular permutation; inserting the insertion sequence into an acceptorsequence, wherein either the insertion sequence or the acceptor sequencecomprises a state; and wherein the respective other sequence isresponsive to a signal; and selecting a fusion molecule, wherein thestate is coupled to the signal, such that the fusion molecule switchesstate in response to the signal.

In some versions of the methods of making fusion molecules the insertionsequence and acceptor sequence can comprise nucleic acids. In thesemethods, insertion includes obtaining a first nucleic acid fragmentencoding an insertion polypeptide and a second nucleic acid fragmentencoding an acceptor polypeptide and inserting the first nucleic acidfragment into the second nucleic acid fragment. In some aspects thismethod is used to provide libraries of fusion nucleic acids encodingfusion polypeptides comprising insertion polypeptides inserted intoacceptor polypeptide sequences. Preferred fusion polypeptides areselected from these libraries in which the states of the insertion andacceptor polypeptides are coupled.

The invention also provides a method for modulating a cellular activity,comprising: providing a fusion molecule generated according to theabove-described methods involving circular permutation of DNA, wherein achange in state of at least the insertion sequence or the acceptorsequence modulates a cellular activity, and wherein the change in statewhich modulates the cellular activity is coupled to a change in state ofthe respective other portion of the fusion molecule. Changing the stateof the respective other portion of the fusion molecule thereby modulatesthe cellular activity.

Yet a further aspect is a method for delivering a bio-effective moleculeto a cell, comprising: providing to the cell a fusion moleculeassociated with a bio-effective molecule generated according to any ofthe above methods, the fusion molecule comprising an insertion sequenceand an acceptor sequence, wherein either the insertion sequence or theacceptor sequence binds to a cellular marker of a pathological conditionand wherein upon binding to the marker, the fusion molecule dissociatesfrom the bio-effective molecule, thereby delivering the molecule to thecell.

Further provided is a method for delivering a bio-effective moleculeintracellularly, comprising: providing to a cell a fusion moleculeassociated with a bio-effective molecule generated according to any ofthe above-described methods involving circular permutation, the fusionmolecule comprising an insertion sequence and an acceptor sequence,wherein either the insertion sequence or acceptor sequence comprises atransport sequence for transporting the fusion molecule intracellularly,and wherein release of the bio-effective molecule from the fusionmolecule is coupled to transport of the fusion molecule intracellularly.

Another aspect of the invention is a method for modulating a molecularpathway in a cell, comprising: providing to a cell a fusion moleculegenerated according to any of the above-described methods, the fusionmolecule comprising an insertion sequence and an acceptor sequence,wherein the activities of the insertion sequence and acceptor sequenceare coupled, and responsive to a signal, and wherein the activity ofeither the insertion sequence or the acceptor sequence modulates theactivity or expression of a molecular pathway molecule in the cell; andexposing the fusion molecule to the signal.

Also provided is a method for controlling the activity of a nucleic acidregulatory sequence, comprising: providing a fusion molecule generatedby circular permutation according to any of the above methods, thefusion molecule comprising an insertion sequence and an acceptorsequence, wherein either the insertion sequence or the acceptor sequenceresponds to a signal, and wherein the respective other sequence of thefusion molecule binds to the nucleic acid regulatory sequence when thesignal is responded to; and exposing the fusion molecule to the signal.

The invention further provides in another aspect a sensor molecule fordetecting a target analyte. The sensor molecule comprises an insertionsequence and an acceptor sequence generated according to any of theabove methods. Either the insertion sequence or the acceptor sequencebinds the analyte, and binding of the analyte is coupled to productionof a signal from the sensor molecule.

In yet another aspect, the invention provides a fusion moleculecomprising: an insertion sequence and an acceptor sequence, generatedaccording to the above-described methods. In one embodiment, either theinsertion sequence or the acceptor sequence transports the fusionmolecule intracellularly, wherein intracellular transport of the fusionmolecule is coupled to binding of the fusion molecule to a bio-effectivemolecule.

Further provided is a fusion molecule generated as described,comprising: an insertion sequence and an acceptor sequence generated bycircular permutation, wherein either the insertion sequence or theacceptor sequence binds to a nucleic acid molecule, and wherein nucleicacid binding activity is coupled to the response of the respective othersequence of the fusion molecule to a signal.

Yet another embodiment is a fusion molecule generated as describedwherein either the insertion sequence or the acceptor sequenceassociates with a bio-effective molecule, and disassociates from thebio-effective molecule, when the respective other sequence of the fusionmolecule binds to a cellular marker of a pathological condition.

Another variation is a fusion molecule capable of switching from anon-toxic to a toxic state, comprising: an insertion sequence and anacceptor sequence generated according to any of the above methodswherein either the insertion sequence or the acceptor sequence binds toa cellular marker of a pathology, and wherein binding of the marker tothe fusion molecule switches the fusion molecule from a non-toxic stateto a toxic state. Other fusion molecules of this type are capable ofswitching from a toxic state to a less toxic state.

The invention further provides “modified” molecular switches generatedaccording to the above methods, wherein as a result of modification, forexample by mutagenesis, the switch is responsive to at least one ligandthat differs from a ligand recognized by an unmodified form of the sameswitch.

Yet a further aspect of the invention is a molecular switch forcontrolling a cellular pathway, comprising: a fusion molecule comprisingan insertion sequence and an acceptor sequence generated according toany of the above methods, wherein the states of the insertion andacceptor sequences are coupled, and responsive to a signal, and whereinthe state of either the insertion sequence or the acceptor sequencemodulates the activity or expression of a molecular pathway molecule ina cell.

Further provided are libraries of molecular switches made according tothe methods of the invention by generating insertion and/or acceptorsequences by circular permutation. The step of insertion can be repeateda plurality of times with a plurality of first and second nucleic acidmolecules, to generate a library of acceptor sequences comprisingcircularized sequences. Preferred library members comprise a firstnucleic acid sequence encoding a first polypeptide having a first state,the first nucleic acid sequence having been circularly permuted andinserted into a second nucleic acid sequence encoding a secondpolypeptide having a second state.

Some versions of the libraries can be produced by iterative processingof at least one existing library, generated according to any of theabove-described methods. In one variation, a selected circularlypermuted insert sequence generated from a first library is inserted intoan acceptor sequence, to generate a second library having a plurality ofmembers, each of which comprise the selected circularly permuted insertsequence. In one embodiment of such a library, the selected circularlypermuted insert sequence is inserted at a random site in the acceptorsequence. In another embodiment, the selected circularly permuted insertsequence is inserted at a non-random site in the acceptor sequence.

The invention further provides isolated nucleic acids encoding molecularswitch proteins. Preferred nucleic acids comprise nucleotide sequencesselected from any of SEQ ID NOS: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, or an effective fragmentthereof.

Yet another aspect of the invention are molecular switch proteinscomprising an amino acid sequence selected from any of SEQ ID NOS: 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, and 75, or an effective fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention can be better understood withreference to the following detailed description and accompanyingdrawings.

FIG. 1 is a schematic diagram illustrating two strategies using circularpermutation and domain insertion for generating libraries of molecularswitches according to the invention. Figure discloses “GSGGG” as SEQ IDNO: 1.

FIG. 2 illustrates steps in creating a cyclized gene using a DKS linkeraccording to the invention. Figure discloses SEQ ID NOS 78-81, 83, 82,and 84, respectively, in order of appearance.

FIG. 3 illustrates steps in creating a cyclized gene using a GSGGGlinker (SEQ ID NO: 1) according to the invention. Figure discloses DNAsequences as SEQ ID NOS 85-86, 87-88, 90, 89, and 91, respectively, inorder of appearance.

FIG. 4 is a diagram illustrating steps in preparing an acceptor DNAsequence for insertion of an insertion DNA sequence at a specific sitein the acceptor DNA sequence according to the invention. Figurediscloses SEQ ID NOS 92-94, 96, 95, 97, and 94, respectively, in orderof appearance.

FIGS. 5A-G are schematic diagrams depicting several applications of themolecular switches of the invention. Figure discloses nucleotides 1-7and 12-21 of SEQ ID NO: 98 and SEQ ID NO: 98, respectively, in order ofappearance.

FIGS. 6A-C illustrate a novel fusion molecule comprising sequences froman effector protein (maltose binding protein, MBP) and an enzyme(β-lactamase, BLA) according to an aspect the invention. FIG. 6A showsthe steps involved in creating the fusion molecule. FIG. 6B is aschematic diagram illustrating the amino acid sequence of the fusionprotein, termed RG13. FIG. 6C is a drawing illustrating the structure ofthe RG13 fusion protein. FIGS. 6B and 6C disclose “GSGGG” as SEQ ID NO:1.

FIGS. 7A-C are three graphs demonstrating characteristics of switchactivity of RG13, a model molecular switch of the invention. FIG. 7Ashows that enzyme activity (nitrocefin hydrolysis) is specific toligands of MBP. FIG. 7B shows reversible switching using competingligand. FIG. 7C shows reversible switching after dialysis.

FIG. 8 is a schematic diagram illustrating coupling of ligand andsubstrate binding.

FIGS. 9A-D show comparisons of characteristics of molecular switchesaccording to the invention. FIG. 9A shows dissociation constants formaltose as a function of apo MBP closure angle. FIGS. 9B-D showsteady-state kinetic parameters of nitrocefin hydrolysis of themolecular switches.

FIG. 10 is a graph showing velocity of nitrocefin hydrolysis by amolecular switch according to the invention as a function of effector(maltose) concentration.

FIG. 11 is a schematic diagram illustrating a strategy for creating alibrary in which a circularly permuted bla gene is inserted into aspecific location in the gene for MBP, according to an embodiment of theinvention. Figure discloses “GSGGG” as SEQ ID NO: 1.

FIG. 12 is a schematic diagram illustrating a strategy for creating alibrary in which a specific circularly permuted version of the bla geneis randomly inserted into a plasmid containing the gene for MBP,according to an embodiment of the invention.

FIGS. 13A and B depict a schematic diagram illustrating constructionschemes and structures of switches isolated from libraries constructedaccording to the invention. Figure discloses “GSGGG” as SEQ ID NO: 1 and“EVKSTS” as SEQ ID NO: 100.

FIGS. 14A-D are four graphs showing enzymatic characteristics ofparticular embodiments of molecular switches according to the invention.

FIG. 15 is a schematic diagram depicting strategies for creating a novelswitch from an existing switch that responds to a particular signalmolecule (in this case maltose).

DETAILED DESCRIPTION

The invention provides improved molecular switches that couple externalsignals to functionality, methods of making these molecules involvingcircular permutation of nucleic acid and amino acid sequences, andmethods of using the same. The switches according to the invention canbe used, for example, to regulate gene transcription, target drugdelivery to specific cells, transport drugs intracellularly, controldrug release, provide conditionally active proteins, perform metabolicengineering, and modulate cell signaling pathways. Libraries comprisingthe switches generated by circular permutation, and expression vectorsand host cells for expressing the switches are also provided.

DEFINITIONS

The following definitions are provided for specific terms which are usedin the following written description.

As used herein, a “molecular switch” refers to a molecule whichgenerates a measurable change in state in response to a signal. In oneaspect, a molecular switch is capable of switching from at least onestate to at least one other state in response to the signal. Preferably,when a portion of the molecule responds to the signal, the portionbecome activated (i.e., turns “ON”) or inactivated (i.e., turns “OFF”).In response to this change in state, the state of another portion of thefusion molecule will change (e.g., turn ON or OFF). In one aspect, aswitch molecule turns ON one portion of the molecule when anotherportion is turned OFF. In another aspect, the switch turns ON oneportion of the molecule, when the other portion is turned ON. In stillanother aspect, the switch molecule turns OFF one portion of themolecule when the other portion is turned ON. In a further aspect, theswitch molecule turns OFF when the other portion is turned OFF.

In some aspects of the invention, a molecular switch exists in more thantwo states, i.e., not simply ON or OFF. For example, a portion of thefusion molecule may display a series of states (e.g., responding todifferent levels of signal), while another portion of the fusionmolecule responds at each state, with a change in one or more states. Amolecular switch also can comprise a plurality of fusion moleculesresponsive to a signal and which mediate a function by changing thestate of at least a portion of the molecule (preferably, in response toa change in state of another portion of the molecule). While the statesof individual fusion molecules in the population may be ON or OFF, theaggregate population of molecules may not be able to mediate thefunction unless a threshold number of molecules switch states. Thus, the“state” of the population of molecules may be somewhere in between ON orOFF depending on the number of molecules which have switched states. Inone aspect, a molecular switch comprises a heterogeneous population offusion molecules comprising members which switch states upon exposure todifferent levels of signal. In other aspects of the invention, however,the state of a single molecule may be somewhere in between ON or OFF.For example, a molecule may comprise a given level of activity, abilityto bind, etc., in one state which is switched to another given level ofactivity, ability to bind, etc., in another state (i.e., an activity,ability to bind, etc., measurably higher or lower than the activity,ability to bind, etc., observed in the previous state).

As used herein, a “state” refers to a condition of being. For example, a“state of a molecule” or a “state of a portion of a molecule” can be aconformation, binding affinity, or activity (e.g., including, but notlimited to, ability to catalyze a substrate; ability to emit light,transfer electrons, transport or localize a molecule, modulatetranscription, translation, replication, supercoiling, and the like).

As defined herein, a molecule, or portion thereof, whose state is“activated” refers to a molecule or portion thereof which performs anactivity, such as catalyzing a substrate, emitting light, transferringelectrons, transporting or localizing a molecule; changing conformation;binding to a molecule, etc.

As defined herein, a molecule, or portion thereof, whose state is“inactivated” refers to a molecule or portion thereof which is, at leasttemporarily, unable to perform an activity or exist in a particularstate (e.g., bind to a molecule, change conformation, etc.).

As used herein, “coupled” refers to a state which is dependent onanother state such that a measurable change in the other state isobserved. As used herein, “measurable” refers to a state that issignificantly different from a baseline or a previously existing stateas determined in a suitable assay using routine statistical methods(e.g., setting p<0.05).

As used herein, “a signal” refers to a molecule or condition that causesa reaction. Signals include, but are not limited to, the presence,absence, or level, of molecules (nucleic acids, proteins, peptides,organic molecules, small molecules), ligands, metabolites, ions,organelles, cell membranes, cells, organisms (e.g., pathogens), and thelike; as well as the presence, absence, or level of chemical, optical,magnetic, or electrical conditions, and can include conditions such asdegrees of temperature and/or pressure. A chemical condition can includea level of ions, e.g., pH.

As used herein, “responsive to a signal” refers to a molecule whosestate is coupled to the presence, absence, or level of the signal.

As used herein, “an insertion sequence” refers to a polymeric sequencewhich is contained within another polymeric sequence (e.g., an “acceptorsequence”) and which conditionally alters the state of the otherpolymeric sequence. An insertion sequence or acceptor sequence cancomprise a polypeptide sequence, nucleic acid sequence (DNA sequence,aptamer sequence, RNA sequence, ribozyme sequence, hybrid sequence,modified or analogous nucleic acid sequence, etc.), carbohydratesequence, and the like. Nucleic acid and amino acid sequences for use asacceptor and insertion sequences in the invention can be naturallyoccurring sequences, engineered sequences (for example, modified naturalsequences), or sequences designed de novo.

As used herein, an “effective fragment” of a nucleic acid or amino acidsequence can include any portion of a full length sequence useful in amolecular switch that has at least 80% of the functional activity of thecorresponding full-length sequence, preferably at least about 90% andmore preferably at least about 95% of that function. By an “effectivefragment” of a molecular switch or related phrase is meant a portion ofa molecular switch protein, or a nucleic acid encoding the same, thathas at least 80% of the activity of the corresponding full-lengthprotein or nucleic acid, determined by an appropriate assay for activityof the particular molecular switch.

As used herein, “multistable” refers to a fusion molecule which iscapable of existing in at least two states.

As used herein, “bistable” refers to a fusion molecule capable ofexisting in two states.

As used herein, “range of states” refers to a series of states in whicha fusion molecule can exist. For example, a range of states can comprisea range of binding activities, a range of light-emitting activities, arange of catalysis efficiencies, and the like.

As used herein, “a change in state” refers to a measurable difference ina state of being of a molecule, as determined by an assay appropriatefor that state.

As used herein, “a graded response” refers to the ability of a fusionmolecule to switch to a series of states in response to a particularthreshold signal.

As used herein, “modulates” or “modulated” or “modulatable” refers to ameasurable change in a state or activity or function. Preferably, wherean activity is being described, “modulated” refers to an at least2-fold, at least 5-fold, at least 10-fold, at least 20-fold or higher,increase or decrease in activity, or an at least 10%, at least 20%, atleast 30%, at least 40% or at least 50% increase or decrease inactivity. However, more generally, any difference which is measurableand statistically different from a baseline is encompassed within theterm “modulated.”

As used herein, a “less active state” is a state which is at least about2-fold less active compared to a given reference state as measured usingan assay suitable for measuring that state, or about at least 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90% or at least about 100% less active. More generally, anydecrease which is measurable and statistically different from baselineis encompassed within the term “less active state.”

As used herein, a “less toxic state” refers to a measurable increase inthe LD₅₀ (i.e., lethal dose which has a 50% probability of causingdeath) or LC₅₀ (i.e., lethal concentration which has a 50% probabilityof causing death). Preferably, a less toxic state is one which isassociated with an at least about 10% increase, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or atleast about 100% increase in LD₅₀ or LC₅₀.

As used herein, “a bio-effective molecule” refers to bioactive moleculewhich can have an effect on the physiology of a cell or which can beused to image a cell. In one aspect, a “bio-effective molecule” is apharmaceutical agent or drug or other material that has a therapeuticeffect on the cell.

As used herein, “a cellular marker of a pathological condition” refersto a molecule which is associated with a cell, e.g., intracellularly orextracellularly, and whose presence or level correlates with thepresence of the disease, i.e., the marker is found in, or on, cells, oris secreted by cells, exhibiting the pathology at levels which aresignificantly different than observed for cells not exhibiting thepathology

As used herein, “a molecular pathway molecule” refers to a moleculewhose activity and/or expression affects the activity and/or expressionof at least two other molecules. Preferably, a molecular pathwaymolecule is a molecule involved in a metabolic or signal transductionpathway. A pathway molecule can comprise a protein, polypeptide,peptide, small molecule, ion, cofactor, organic and inorganic molecule,and the like.

As used herein, “modulating a molecular pathway” refers to a change inthe expression and/or activity of at least one pathway molecule.

As used herein, “at an insertion site” of a nucleic acid molecule refersto from about 1 to 21 nucleotides immediately flanking the insertionsite.

As used herein, “randomly inserting” refers to insertion at non-selectedsites in a polymeric sequence. In one aspect, “random insertion” refersto insertion that occurs in a substantially non-biased fashion, i.e.,there is a substantially equal probability of inserting between membersof any pairs of monomers (e.g., nucleotides or amino acids) in anacceptor molecule comprising a given number of monomeric sequences.However, in another aspect, random insertion has some degree of bias,e.g., there is a greater than equal probability of inserting atdifferent sites. Minimally, the probability of insertion at a site in anacceptor sequence is greater than zero but less than one.

As used herein, “a new activity” refers to an activity which is notfound in either donor or acceptor sequences. Generally, fusion moleculesaccording to the invention comprise a new activity in that the activityof the acceptor sequence or insertion sequence is newly coupled to thestate of the respective other portion of the sequence. An insertion oracceptor sequence also may comprise a catalytic site which responds to(e.g., catalyzes) a substrate provided in the form of the respectiveother portion of the fusion molecule, thereby producing a fusionmolecule which comprises an activity present in neither the originalcatalytic site or the substrate (e.g., such as the ability toself-cleave in the presence of a signal).

As used herein, “a nuclear regulatory sequence” refers to a nucleic acidsequence which is capable of modulating the activity of another nucleicacid in cis or in trans. Types of activities regulated include, but arenot limited to, modulating transcription, translation, replication,recombination, or supercoiling. A nucleic acid regulatory sequence caninclude promoter elements, operator elements, repressor elements,enhancer sequences, ribosome binding sites, IRES sequences, origins ofreplication, recombination hotspots, topoisomerase binding sequences,and the like.

As used herein, “altered by bisection” refers to a change in state uponfragmenting a polypeptide into two pieces. The term “bisection” does notimply that the polypeptide is divided into fragments of equal size;rather fragments can be generated by cleaving anywhere along the lengthof the primary sequence of the amino acid.

As used herein, “selecting for restoration of function or state” refersto selection for restoration of a function or state which issufficiently similar to that of the original function under assayconditions suitable for evaluating the function or state. As usedherein, “sufficiently similar” refers to a state that can achieve theoriginal function in an effective manner. For example, when thefunction/state is binding, restoration of function/state can beevaluated by generating Scatchard plots and/or determining K_(d). Whenthe function/state is the ability of a molecule to generate light,restoration can be measured spectrophotometrically, for example.

As used herein, a “modification” of a polypeptide refers to an addition,substitution or deletion of one or more amino acids in a polypeptidewhich does not substantially alter the state of the polypeptide. Forexample, where a state is an activity of a polypeptide, a modificationresults in no more than a 10% decrease or increase in the activity ofthe polypeptide, and preferably no more than a 5% decrease or increasein the activity of the polypeptide.

As used herein, the terms “cyclization,” and “cyclized” in respect to anucleic acid or protein sequence or fragment thereof, refer to theprocess of taking a non-cyclized sequence of nucleic acid or amino acidand converting it to a cyclized form. For example, a “cyclized” nucleicacid is a form of nucleic acid in which every nucleotide in the nucleicacid sequence is covalently bonded to exactly two other nucleotides,typically through phosphate bridges between the 3′ and 5′ positions ofthe sugar residue of the nucleotide. This is distinguished from a“linear” form of a nucleic acid sequence in which the nucleotides on the5′ and 3′ ends are attached to only one nucleotide. In a cyclized formof an amino acid sequence, the N- and C-termini are fused generallythrough a linker sequence. If the original N- and C-termini are proximalto one other, generally a shorter linker is used than if they arefarther apart.

As used herein, the term “circularly permuted” refers to a nucleic acidor protein sequence in which the primary sequence differs from theoriginal non-circularly permuted sequence in a specific way. For anucleic acid, the circularly permuted sequence differs in that acontinuous sequence that was on the 3′ end in the non-circularlypermuted sequence is attached to the 5′ end in the circularly permutedsequence. The circularly permuted nucleic acid may or may not have alinker sequence between the original 5′ and 3′ ends. For a protein, thecircularly permuted sequence differs in that a continuous sequence thatwas on the C-terminus in the non-circularly permuted sequence isattached to the N-terminus in the circularly permuted sequence. Thecircularly permuted protein may or may not have a linker sequencebetween the original N- and C-termini A circularly permuted sequence canbe conceptualized as joining the ends of an original, linearnon-circularly permuted sequence to form a cyclized sequence, andconverting the cyclized sequence back to a linear sequence by breakingthe bonds at a new location. Although a circularly permuted sequence canbe created in this manner, as used herein, the term “circularly permutedsequence” can also include the same sequence created by other means notinvolving a cyclized intermediate. “Randomly circularly permuted” asused herein refers to a sequence in which a circularly permuted sequenceis created in which the site of circular permutation is determined by arandom, semi-random or stochastic process.

Generating Fusion Molecules Using Random Circular Permutation

In one aspect, the invention includes a method for assembling a fusionmolecule comprising randomly circularly permuting an insertion sequenceand inserting the insertion sequence into an acceptor sequence.Exemplary insertion and acceptor sequences including known “domain”sequences that can be combined to form fusion molecules are discussed infurther detail infra, and generally include any two sequences desired tobe functionally combined in a fusion molecule to form a molecularswitch.

By using a combinatorial approach, a plurality of potential switches iscreated from which to select switches with optimized characteristics.This method is advantageous over existing domain insertion methods inthat vastly increased numbers of geometric configurations between theacceptor sequence and the insertion sequence can be generated and madeavailable for testing. As discussed, the switching behavior achieved todate by existing methods is generally modest (i.e., less than about2-fold effect). See, for example, PCT Publication WO 03/078575, hereinincorporated by reference, and Guntas and Ostermeier (2004). As shown inExamples herein, the invention provides significantly improved molecularswitches, for example with switching activity up to at least about35-fold, and modified switches that respond to novel effector molecules.

A number of different strategies can be used to create the fusionmolecules of the instant invention. FIG. 1 shows two preferredstrategies for creating molecular switches using random circularpermutation of DNA in combination with domain insertion. The strategiesare generally applicable to creating any desired molecular switches, andare illustrated in FIG. 1 and several Examples herein, using exemplaryfusions that combine sequences from two non-homologous proteins, in thiscase an enzyme (i.e., E. coli TEM β-lactamase, BLA) with sequences froman effector or signal protein (in this case, E. coli maltose bindingprotein, MBP) that responds to a signal (i.e., maltose). As shown below,the BLA-MBP fusion proteins produced by the methods of the invention canact as molecular switches, for example by functioning as BLA enzymesonly in the presence of maltose.

Preparing Circularly Permuted Insert Genes

Referring to FIG. 1, circular permutation of at least one of the genes(in this case the insert gene) is central to the method. Althoughcircular permutation of the insert gene is shown, circular permutationof the acceptor sequence, or both sequences, is within the invention. Inthe example shown in FIG. 1, BLA is the insert gene, and MBP is theacceptor gene.

As is known in the art, a circularly permuted protein has its originalN- and C-termini fused and new N- and C-termini created by a breakelsewhere in the sequence. The insert gene is circularly permutatedusing any suitable technique. Exemplary techniques for circularpermutation by chemical or genetic methods include but are not limitedto those described for example by Goldenberg and Creighton (1983), andHeinemann and Hahn (1995). A particularly preferred genetic method forrandom circular permutation is that of Graf and Schachmann (1996). Seealso Ostermeier and Benkovic (2001).

Referring to the central portion of FIG. 1, a preferred method ofrandomly circularly permuting a sequence can generally include thefollowing steps:

(i) isolating a linear fragment of double-stranded DNA of the gene to berandomly circularly permuted with a linker sequence and flankingcompatible ends;

(ii) cyclizing the DNA fragment by ligation under dilute conditions;

(iii) randomly linearizing the cyclized gene, for example usingdigestion by a nuclease such as DNaseI under conditions in which theenzyme, on average, makes one double-strand break;

(iv) repairing nicks and gaps, for example using enzymes such as DNApolymerase and DNA ligase, respectively; and

(v) ligating the fragment into a desired vector comprising the acceptorsequence by blunt end ligation, to create a library of randomlycircularly permuted sequences.

Preferred methods for preparing cyclized genes include a step of addingDNA that codes for a “linker” to link the original N- and C-termini. Anysuitable linker sequences can be used for this purpose. Preferredmethods of cyclizing a gene utilize linkers such a “DKS linker” (Osunaet al., 2002) or a flexible pentapeptide linker such as a “GSGGG linker”(SEQ ID NO: 1) having the amino acid sequence GSGGG (SEQ ID NO: 1). Seealso Example 1, infra, for further details. Generally, the gene fragmentof interest (for example a fragment encoding a selected amino acidsequence, such as amino acids 24-286 of the β-lactamase protein), isamplified by a suitable technique such as polymerase chain reaction(PCR) under conditions resulting in flanking of the selected sequence byrestriction enzyme site sequences coding for the linkers, and is thencloned into a suitable vector such as pGem T-vector (Promega). Exemplarycloning vectors containing the sequences comprising linkers areindicated in FIG. 1 as pBLA-CP(DKS) or pBLA-CP(GSGGG) (“GSGGG” disclosedas SEQ ID NO: 1).

The fragments to be cyclized are then released from the cloning vectorby digestion with a suitable restriction enzyme and purified, forexample by agarose gel electrophoresis. Cyclizing is achieved, forexample, by treating with a ligase such as T4 DNA ligase. The cyclized(circular) fragments are subsequently purified and subjected to circularpermutation (step iii above). Exemplary circularized genes comprisingDKS and GSGGG (SEQ ID NO: 1) linkers according to the invention areshown in FIGS. 2 and 3, respectively.

Referring again to FIG. 1, the circularized genes are randomlylinearized, by subjecting them to cleavage with a digestion enzyme thatmakes on average one double-strand break in the circularized DNA. Apreferred enzyme for use in this step is a nuclease. A particularlypreferred enzyme is DNaseI. The conditions for nuclease digestion can bedetermined experimentally by varying the amount of enzyme added andanalyzing the digested products by agarose gel electrophoresis.Generally, approximately 1 milliunit of DNaseI per microgram of DNA (ata concentration of 10 micrograms per ml) for an 8-minute digestion at22° C. is suitable, but will vary somewhat for each library. See alsoExample 1 for further details of suitable conditions for the digestionstep. In addition to digestion by nucleases (e.g., DNAse, S1,exonucleases, restriction endonucleases and the like), other methods forintroducing breaks in sequences can be used. For example, mechanicalshearing, chemical treatment, and/or radiation can be used. Generally,the method for introducing breaks is not intended to be limiting.

Libraries Comprising Circularly Permuted Insert Sequences

In one aspect, libraries comprising a plurality of library members areprovided by the invention. Each library member comprises a first nucleicacid sequence encoding a first polypeptide having a first state, thefirst nucleic acid sequence having been randomly circularly permuted andinserted into a second nucleic acid encoding a second polypeptide havinga second state. The libraries can be constructed in any suitable mannerknown in the art of molecular biology.

In one preferred type of library, the randomly circularly permutedsequences are randomly inserted into acceptor sequences, a strategywhich maximizes the number of possible combinations of insertion andacceptor sequences. Several different strategies can be used to makesuch “random insertion” libraries. One preferred embodiment of themethod, i.e., “Circular Permutation of Insert and Random DomainInsertion,” is shown on the left side of FIG. 1. In this embodiment, thecircularly permuted insertion sequence is inserted at a random site in avector, such as a plasmid, comprising the acceptor sequence. Invariations of this method, (both shown in FIG. 1), entire libraries ofcircularly permuted insert sequences can be randomly inserted into theacceptor sequences, or specific circularly permuted versions of aselected sequence can be randomly inserted into the vector. (See, forexample, FIG. 12.) See also Example 5 and FIG. 13 showing variousstrategies including iterative approaches for constructing librariesusing circularly permuted DNA, including selected preferred sequencespreviously generated by circular permutation according to the invention.See, for example, the descriptions of Libraries 6 and 7 in Example 5.

Preparing Target (Acceptor) DNA for Random Insertion Libraries

As discussed, in one aspect, libraries are constructed in which aninsertion sequence has been randomly inserted into an acceptor sequence.Preferably, such libraries are generated by randomly inserting a nucleicacid fragment encoding an insertion sequence into a nucleic acidfragment encoding an acceptor sequence.

Existing methods for random insertion can be categorized into one of twostrategies: insertion via transposons and insertion after a randomdouble stranded break in DNA using one or a combination of nucleases. Avariety of transposons have been used to deliver short, in-frameinsertions of 4-93 amino acids (e.g., Hayes and Hallet, 2000, TrendsMicrobiol. 8: 571-7; and Manoil and Traxler, 2000, Methods 20: 55-61).However, although transposons are an efficient method for delivering aninsertion, insertion methods are preferred which create libraries withdirect insertions, deletions at the insertion site, or variability inthe amount of deletions or tandem duplication or variability in thedistribution of direct insertions, deletions and tandem duplications.

Random insertion using nuclease treatment, on the other hand, can createsuch libraries. These methods typically are used for the insertion ofshort sequences into a target gene for example during linker scanningmutagenesis. These methods generally differ in the strategy used toproduce a random, double-strand break in supercoiled plasmid DNAcontaining the gene to be inserted.

Any suitable procedure for randomly inserting a first sequence intosecond sequence can be used. Exemplary methods are described, forexample, in PCT Publication WO 03/078575, herein incorporated byreference. As discussed, the use of BLA and MBP as respective insertionand acceptor sequences, and the use of particular vectors are merelyexemplary; potentially any two proteins can be functionally coupled inthis manner following random circular permutation of one or bothsequences.

To prepare a random insertion library, a target vector comprising thenucleic acid encoding the acceptor polypeptide is preferably randomlylinearized (see FIG. 1, left side). For linearization, a variety ofdifferent nucleases and digestion schemes can be used. For example, thevector may be exposed to DNase/Mn²⁺ digestion followed bypolymerase/ligase repair; S1 nuclease digestion followed bypolymerase/ligase repair; or S1 nuclease digestion which is notrepaired. The three schemes differ in (a) the methods used to create therandom double-stranded break in the target plasmid and (b) whether ornot the nucleic acid (e.g., DNA) is repaired by polymerase/ligasetreatment, or other methods. However, it should be apparent to those ofskill in the art that any method of introducing breaks into a DNAmolecule can be used (e.g., such as digestion by mung bean nucleases,endonucleases, restriction enzymes, exposure to chemical agents,irradiation, and/or mechanical shearing) and that the methods ofintroducing breaks described above are not intended to be limiting.

Preferably, digestion is controlled such that a significant fraction ofDNA is undigested in order maximize the amount of linear DNA that hasonly one double strand break. Key features for optimizing DNase Idigestion include the use of Mg²⁺ free DNaseI (Roche MolecularBiochemicals), a digestion temperature of 22° C. and 1 mM Mn²⁺ insteadof Mg²⁺ to increase the ratio of double strand breaks to nicks (see,e.g., as described in Campbell and Jackson, 1980, J. Biol. Chem. 255:3726-35).

The DNA can be repaired using methods known in the art, for example,using T4 DNA ligase and T4 DNA polymerase (see, e.g., Graf andSchachman, 1996, Proc. Natl. Acad. Sci. USA 93: 11591-11596), anddephosphorylated. Ligation with nucleic acids encoding the insert isperformed and nucleic acids (e.g., library members) are collected.

Preparing Target (Acceptor) DNA for Site-Specific Insertion Libraries

Referring again to FIG. 1, another aspect of the invention is shown onthe right side of the Figure (“Random Circular Permutation of Insert andDomain Insertion at a Specific Site). In this approach, the circularlypermuted insertion sequence is inserted into a selected site in theacceptor sequence. Any suitable site can be selected in the acceptorsequence, based upon desired functional outcome and knowledge of thestructure of the acceptor sequence. For example, this site could be asite previously shown to be useful for creating molecular switches (asis demonstrated in Examples below) or a site that is predicted, bycomputational methods or other means, to be useful in creating amolecular switch.

For insertion at a specific site, plasmids comprising insertionsequences can be modified as shown in FIG. 4, for example by insertionof inverted SapI sites between particular bases such that digestion withSapI and subsequent filling in of the resulting overhangs using Klenowpolymerase in the presence of dNTPs results in a bisected perfectlyblunt sequence on one side (e.g., MBP [1-165]) and a perfectly bluntsequence (e.g., MBP [164-370]) on the other side. SapI is a type IISrestriction enzyme that cuts outside its recognition sequence. Othertype IIS restriction enzymes can also be used, as well as non-type IISrestriction enzymes. The randomly permuted insert sequence issubsequently inserted into the acceptor sequence at the selected site(FIGS. 1 and 11). See also Examples 1 and 4, supra.

Target Vectors Comprising Acceptor Sequences

In one aspect, construction of a library comprises the initial step ofconstructing and testing a target vector, i.e., a vector comprising anucleic acid encoding an acceptor sequence. For example, a gene or genefragment which encodes a polypeptide is cloned into a vector, such as aplasmid. Preferably, the polypeptide exists in a state at least undercertain conditions, i.e., comprises an activity, can bind a molecule,exist in a conformation, emit light, transfer electrons, catalyze asubstrate, etc. under those conditions.

Preferably, the plasmid comprises a reporter sequence for monitoring theefficacy of the cloning process. Suitable reporter genes include anygenes that express a detectable gene product which may be RNA orprotein. Examples of reporter genes, include, but are not limited to:CAT (chloramphenicol acetyl transferase); luciferase, and other enzymedetection systems, such as β-galactosidase, firefly luciferase,bacterial luciferase, phycobiliproteins (e.g., phycoerythrin); GFP;alkaline phosphatase; and genes encoding proteins conferringdrug/antibiotic resistance, or which encode proteins required tocomplement an auxotrophic phenotype. Other useful reporter genes encodecell surface proteins for which antibodies or ligands are available.Expression of the reporter gene allows cells to be detected or affinitypurified by the presence of the surface protein.

The reporter gene also may be a fusion gene that includes a desiredtranscriptional regulatory sequence, for example, to select for a fusionmolecule whose switching functions include the ability to modulatetranscription.

Vectors for Expressing Fusion Molecules

Identification of desired fusion molecules, whether created by random orsite-specific insertions, can be facilitated by the use of expressionvectors in creating the libraries described above. Such expressionvectors additionally can be useful for generating large amounts offusion molecules (e.g., for delivery to a cell, or organism, for use invitro or in vivo).

Thus, in one aspect, library members comprise regulatory sequences(e.g., such as promoter sequences) which can be either constitutivelyactive or inducible which are operatively linked to acceptor sequencescomprising insertion sequences. Regulatory sequences can comprisepromoters and/or enhancer regions from a single gene or can combineregulatory elements of more than one gene. In a preferred embodiment,the regulatory sequences comprise strong promoters which allow highexpression in cells, particularly in mammalian cells. For example, thepromoter can comprise a CMV promoter and/or a Tet regulatory element.

Library members also can comprise promoters to facilitate in vitrotranslation (e.g., T7, T4, or SP6 promoters). Such constructs can beused to produce amounts of fusion molecules in sufficient quantity toverify initial screening results (e.g., the ability of the molecules tofunction as molecular switches).

The expression vectors can be self-replicating extrachromosomal vectorsand/or vectors which integrate into a host genome. In one aspect, theexpression vectors are designed to have at least two replicationsystems, allowing them to be replicated and/or expressed and/orintegrated in more than one host cell (e.g., a prokaryotic, yeast,insect, and/or mammalian cell). For example, the expression vectors canbe replicated and maintained in a prokaryotic cell and then transferred(e.g., by transfection, transformation, electroporation, microinjection,cell fusion, and the like) to a mammalian cell.

The expression vectors can include sequences which facilitateintegration into a host genome (e.g., such as a mammalian cell). Forexample, the expression vector can comprise two homologous sequencesflanking the nucleic acid sequence encoding the fusion molecule,facilitating insertion of the nucleic acid expressing the fusionmolecule into the host genome through recombination between the flankingsequences and sequences in the host genome. Sequences such as lox-cresites also can be provided for tissue-specific inversion of the fusionmolecule nucleic acid with respect to a regulatory sequence to which thefusion molecule nucleic acid is operably linked.

Integration into the host genome may be monitored by screening for theexpression of a reporter sequence included in the expression vector, bythe expression of the unique fusion molecule (e.g., by monitoringtranscription via Northern blot analysis or translation by animmunoassay), and/or by the presence of the switching activity in thecell.

Evaluating Libraries for Identification of Fusion Molecules

In one aspect, transformants are selected which express a reporter geneincluded in the target vector, such as a drug resistance gene toinitially screen for fusion molecules. Alternatively, or additionally,transformants can be selected in which the state of the insertionsequence is coupled to the state of the acceptor sequence. Thus, in oneaspect, the existence of each state is assayed for, as is the dependenceof each state on the existence of one or more other states. States maybe assayed for simultaneously, or sequentially, in the same host cell orin clones of host cells. Fusion molecules also can be isolated from hostcells (or clones thereof) and their states can be assayed for in vitro.

For example, in one aspect, the enzymatic activity of an insertionsequence or acceptor sequence is assayed for at the same time that thebinding activity of the respective other portion of the fusion isevaluated to identify fusion molecules in which enzymatic activity isdependent on binding activity.

In another aspect, libraries are screened for fusion molecules whichbind to a molecule, such as a bio-effective molecule (e.g., a drug,therapeutic agent, toxic agent, or agent for affecting cellularphysiology). The bound fusion molecule is exposed to a cell, and theability of the fusion molecule to be localized intracellularly isdetermined. Preferably, release of the bio-effective molecule inresponse to intracellular localization also is determined.

For example, a cell can be transiently permeabilized (e.g., by exposureto a chemical agent such as Ca²⁺ or by electroporation) and exposed to afusion molecule associated with the bio-effective molecule (e.g., boundto the bio-effective molecule), allowing the fusion molecule and boundmolecule to gain entry into the cell. The ability of the fusion moleculeto localize to an intracellular compartment (e.g., to the endoplasmicreticulum, to a lysosomal compartment, nucleus, etc.) along with thebio-effective molecule can be monitored through the presence of a label(e.g., such as a fluorescent label or radioactive label) on the fusionmolecule, bio-effective molecule, or both. The label can be conjugatedto the fusion molecule and/or the bio-effective molecule using routinechemical methods known in the art. A label also may be provided as partof an additional domain of the fusion molecule. For example, the fusionmolecule can comprise a GFP polypeptide or modified form thereof. Thelocalization of the label (and hence the fusion molecule and/orbio-effective molecule) can be determined for example using lightmicroscopy. Release of the bio-effective molecule can be monitored bylysing the cell, immunoprecipitating the fusion molecule, and detectingthe amount of labeled bio-effective molecule in the precipitatedfraction.

In one aspect, the cell need not be permeabilized to allow entry of thefusion molecule because the fusion molecule comprises a signal sequencethat enables the fusion molecule to traverse the cell membrane.Intracellular transport of the bio-effective molecule can be monitoredby labeling the bio-effective molecule and examining its localizationusing light microscopy, FACs analysis, or other methods routine in theart.

In another aspect, insertion libraries are screened for fusion moleculeswhich comprise an insertion sequence or acceptor sequence whichassociates with a bio-effective molecule and which releases thebio-effective molecule when the respective other portion of the fusionmolecule binds to a cellular marker of a pathological condition. Thus,in one aspect, fusion molecules associated with a bio-effective moleculeare contacted with cells expressing such a marker and the ability of thefusion molecules to specifically bind to the cell is assayed for, aswell as the ability of the fusion molecule to release the bio-effectivemolecule in response to such binding. For example, as above, either, orboth, the fusion molecule and the bio-effective molecule can be labeledand the localization of the molecules determined. The action of thebio-effective molecule also can be monitored (e.g., the effect of thebio-effective molecule on the cell can be monitored).

In still another aspect, insertion libraries are screened for fusionmolecules which can switch from a non-toxic state to a toxic state uponbinding of the insertion sequence or acceptor sequence to a cellularmarker of a pathology. Fusion molecules can be selected whichspecifically bind to cells expressing the marker, and the effect of thefusion molecules on cell death can be assessed. Cell death can bemonitored using methods routine in the art, including, but not limitedto: staining cells with vital dyes, detecting spectral propertiescharacteristic of dead or dying cells, evaluating the morphology of thecells, examining DNA fragmentation, detecting the presence of proteinsassociated with cell death, and the like. Cell death also can beevaluated by determining the LD₅₀ or LC₅₀ of the fusion molecule.

In a further aspect, the insertion library is screened for fusionmolecules which comprise a molecular switch for controlling a cellularpathway. Preferably, the states of the insertion sequence and acceptorsequence in the fusion molecules are coupled and responsive to a signalsuch that in the presence of the signal, the state of either theinsertion sequence or the acceptor sequence modulates the activity orexpression of a molecular pathway molecule in a cell. A signal can bethe presence, absence, or level, of an exogenous or endogenous bindingmolecule to which either the insertion sequence or acceptor sequencebinds, or it can be a condition (e.g., chemical, optical, electrical,etc.) in an environment to which the fusion molecule is exposed. Theability of the fusion molecule to control a pathway can be monitored byexamining the expression and/or activity of pathway molecules which actdownstream of a pathway molecule whose expression and/or activity isbeing modulated.

In another aspect, fusion molecules are selected in which either theinsertion sequence or acceptor sequence binds to a nucleic acidmolecule. For example, the ability of the fusion molecules to bind to anucleic acid immobilized on a solid phase can be monitored (e.g.,membrane, chip, wafer, particle, slide, column, microbead, microsphere,capillary, and the like). Preferably, fusion molecules are selected inwhich nucleic acid binding activity is coupled to a change in state ofthe respective other sequence of the fusion molecule. For example,nucleic acid binding activity can be coupled to the binding activity ofanother portion of the fusion molecule, catalysis by the other portion,the light emitting function of the other portion, electron transferringability of the other portion, ability of the other portion to changeconformation, and the like. Preferably, nucleic acid binding activity iscoupled to the response of the fusion molecule to a signal.

Nucleic acid binding activity also can be monitored by evaluating theactivity of a target nucleic acid sequence to which the fusion moleculebinds. For example, in one aspect, the fusion molecule binds to anucleic acid regulatory sequence which modulates the activity (e.g.,transcription, translation, replication, recombination, supercoiling) ofanother nucleic acid molecule to which the regulatory sequence isoperably linked. The nucleic acid regulatory molecule and its regulatedsequence can be provided as part of a nucleic acid molecule encoding thefusion molecule or can be provided as part of a separate molecule(s).The nucleic acid binding activity can be monitored in vitro or in vivo.The ability of fusion molecules to bind to a nucleic acid can also bedetermined in vivo using one-hybrid or two-hybrid systems (for example,see, Hu, et al., 2000, Methods 20: 80-94).

In certain aspects, fusion molecules are selected which bind to a knownregulatory sequence or a sequence naturally found in a cell. In otheraspects, a sequence which is not known to be a regulatory sequence in acell is selected for. Preferably, such a sequence binds to the fusionmolecule and modulates the activity of another nucleic acid (in cis orin trans). Thus, the fusion molecule can be used to select for novelnucleic acid regulatory sequences. Preferably, the fusion moleculemodulates the regulatory activity of the nucleic acid molecule inresponse to a signal, as described above.

In still a further aspect, the insertion library is screened for fusionmolecules which are sensor molecules. Preferably, fusion molecules arescreened for in which either the insertion sequence or acceptor sequencebinds to a target molecule and wherein the respective other portion ofthe fusion molecule generates a signal in response to binding. Signalscan include: emission of light, transfer of electrons, catalysis of asubstrate, binding to a detectable molecule, and the like. To assay forsuch fusions, members of the library can be screened in the presence ofthe target molecule (e.g., in solution, or immobilized on a solidsupport) for the production of the signal.

Fusion Molecules Comprising Coupled Insertion and Acceptor Sequences

In one aspect, a modulatable fusion molecule is provided which comprisesan insertion sequence and an acceptor sequence which contains theinsertion sequence (Several examples of such fusion molecules are shown,e.g., in FIG. 13). Preferably, the insertion sequence and acceptorsequence are polymeric molecules, e.g., such as polypeptides or nucleicacids. More preferably, both the insertion sequence and acceptorsequence are capable of existing in at least two states and the state ofthe insertion sequence is coupled to the state of the acceptor sequenceupon fusion, such that a change in state in either the insertionsequence or acceptor sequence will result in a change in state of therespective other portion of the fusion. As discussed, a “state” can be aconformation; binding affinity; ability or latent ability to catalyze asubstrate; ability or latent ability to emit light; ability or latentability to transfer electrons; ability or latent ability to withstanddegradation (e.g., by a protease or nuclease); ability or latent abilityto modulate transcription; ability or latent ability to modulatetranslation; ability or latent ability to modulate replication; abilityor latent ability to initiate or mediate recombination or supercoiling;or otherwise perform a function; and the like.

Preferably, the change in state is triggered by a signal to which thefusion molecule is exposed, e.g., such as the presence, absence, oramount of a small molecule, ligand, metabolite, ion, organelle, cellmembrane, cell, organism (e.g., such as a pathogen), temperature change,pressure change, and the like, to which the fusion molecule binds; achange in a condition, such as pH, or a change in the chemical, optical,electrical, or magnetic environment of the fusion molecule. In oneaspect, a fusion molecule functions as an ON/OFF switch in response to asignal (e.g., changing from one state to another). For example, when aninsertion sequence or acceptor sequence of the fusion molecule binds toa ligand, the respective other half of the fusion may change state(e.g., change conformation, bind to a molecule, release a molecule towhich it is bound, catalyze a substrate or stop catalyzing a substrate,emit light or stop emitting light, transfer electrons or stoptransferring electrons, activate or inhibit transcription, translation,replication, etc.).

Some fusion molecules according to the invention also can be used togenerate graded responses. In this scenario, a fusion molecule canswitch from a series of states (e.g., more than two different types ofconformations, levels of activity, degrees of binding, levels of lighttransmission, electron transfer, transcription, translation,replication, etc.). Preferably, the difference in state is one which canbe distinguished readily from other states (e.g., there is a significantmeasurable difference between one state and any other state, asdetermined using assays appropriate for measuring that state).

More generally, a molecular switch can generate a measurable change instate in response to a signal. For example, a molecular switch cancomprise a plurality of fusion molecules each responsive to a signal andfor mediating a function in response to a change in state of at least aportion of the molecule. As above, preferably, this change of stateoccurs in response to a change in the state of another portion of themolecule.

While the states of individual fusion molecules in the population may beON or OFF, the aggregate population of molecules may not be able tomediate the function unless a threshold number of molecules switchstates. Thus, the “state” of the population of molecules may besomewhere in between ON or OFF, depending on the number of moleculeswhich have switched states. This provides an ability to more preciselytune a molecular response to a signal by selecting for molecules whichrespond to a range of signals and modifying the population of fusionmolecules to provide selected numbers of fusion molecules, providing anaggregate switch which can respond to a narrow range or wider range ofsignal as desired. Thus, in one aspect, a heterogeneous population offusion molecules is provided comprising members which respond todifferent levels or ranges of signals. Individual fusion molecules alsomay exist in states intermediate between ON or OFF; e.g., having a givenlevel of activity, ability to bind to a molecule in one state and ameasurably higher or lower level of activity, ability to bind, etc., ina different state.

Insertion Sequences

The size of the insertion in the fusion protein will vary depending onthe size of insertion sequence required to confer a particular state onthe insertion sequence without significantly disrupting the ability ofthe acceptor molecule into which it is inserted to change state.Preferably, the effect of the insertion is to couple the change in stateof the acceptor molecule to a change in state of the insertion molecule,or vice versa.

Generally, for polypeptide insertions, the size of the insertionsequence can range from about two amino acids to at least about 1000,for example at least about 900, 800, 700, 600, 500, 400, 300, 200, 100,or fewer amino acids. In one aspect, the insertion comprises a domainsequence with a known characterized activity (e.g., a portion of aprotein in which bioactivity resides); however, in other aspects, theinsertion sequence comprises sequences up to an entire protein sequence.

Acceptor Sequences

Generally, there are no constraints on the size or type of acceptorsequence which can be used. However, in one aspect, an acceptor sequenceis a polypeptide whose state resides in a discontinuous domain of aprotein (e.g., the amino acids involved in conferring the state/activityof the acceptor sequence are not necessarily contiguous in the primarypolypeptide sequence) (see, e.g., as described in Russell and Ponting,1998, Curr. Opin. Struct. Biol. 8: 364-371, and Jones, et al., 1998,Protein Sci. 7: 233-42).

Suitable polypeptides for acceptor molecules can be identified usingdomain assignment algorithms such as are known in the art (e.g., such asthe PUU, DETECTIVE, DOMAK, and DomainParser, programs). For example, aconsensus approach may be used as described in Jones, et al., (1998).Information also can be obtained from a number of molecular modelingdatabases such as the web-based NIH Molecular Modeling Homepage, or the3Dee Database described by Dengler, et al., 2001, Proteins 42(3):332-44. However, the most important criterion for selecting a sequenceis its function, e.g., the desired state parameters of the fusionmolecule.

However, in a further aspect, no pre-screening is done and an acceptorsequence is selected simply on the basis of a desired activity. Thepower of the methods according to the invention is that they rely oncombinatorial screening to identify any, and preferably, all,combinations of insertions that produce a desired coupling in states ofacceptor and insertion molecules.

Domain Sequences in Fusion Proteins

In one aspect, the insertion sequence or acceptor sequence comprises a“domain” sequence having a known state. Domains can be minimalsequences, such as are known in the art, which are associated with aparticular known state, or can be an entire protein comprising thedomain or a functional fragment thereof.

The insertion and acceptor sequences can be selected from any of thedomain sequences described below and can be of like kind (e.g., bothcatalytic sites, both binding domains, both light emitting domains) orof different kind (e.g., a catalytic site and a binding site, as shownfor example in FIG. 6B, a binding site and a light emitting domain;etc.). The domain sequences can be the minimal sequences required toconfer a state or activity or can comprise additional sequences. Otherinsertion and acceptor sequences can be derived from known domainsequences or from newly identified sequences. Such sequences are alsoencompassed within the scope of the instant invention.

Minimal domain sequences can be defined by site-directed mutagenesis ofa sequence having a desired state to determine the minimum amino acidsnecessary to confer the existence of the state under the appropriateconditions (e.g., such as a minimal binding site sequence or a minimumsequence necessary for catalysis, light emission, etc.). As discussedabove, minimal domain sequences also can be defined virtually, usingalgorithms to identify consensus sequences or areas of likely proteinfolding. Once a domain sequence has been identified, it can be modifiedto include additional sequences, as well as insertions, deletions, andsubstitutions of amino acids so long as they do not substantially affectthe state of the domain sequence. While domain sequences can be obtainedusing nucleic acids encoding appropriate fragments of polypeptides, theyalso can be synthesized, for example, based on a predicted consensussequence for a class of molecules which is associated with a particularstate. However, as discussed above, in some cases it may be desirable toprovide the domain sequence in the form of a native protein comprisingthe domain.

Suitable domain sequences include extracellular domains which areportions of proteins normally found outside of the plasma membrane of acell. Preferably, such domains bind to bio-effective molecules. Forexample, an extracellular domain can include the extracytoplasmicportion of a transmembrane protein, a secreted protein, a cell surfacetargeting protein, a cell adhesion molecule, and the like. In oneaspect, an extracellular domain is a clustering domain, which, uponactivation by a bio-effective molecule will dimerize or oligomerize withother molecules comprising extracellular domains.

Intracellular domains also can serve as insertion sequences or acceptorsequences. As used herein, “an intracellular domain” refers to a portionof a protein which generally resides inside of a cell with respect tothe cellular membrane. In one aspect, an intracellular domain is onewhich transduces an extracellular signal into an intracellular response.For example, an intracellular domain can comprise a proliferation domainwhich signals a cell to enter mitosis (e.g., such as domains from Jakkinase polypeptides, I1-2 receptor β and/or gamma chains, and the like).Other transducer sequences include sequences from the zeta chain of theT cell receptor or any of its homologs (e.g., the eta chain, Fc epsilonR1-gamma and -62 chains, MB1 chain, B29 chain, and the like), CD3polypeptides (gamma, beta and epsilon), syk family tyrosine kinases(Syk, ZAP 70, and the like), and src family tyrosine kinases (Lck, Fyn,Lyn, and the like).

A transmembrane domain also can be used as an insertion sequence oracceptor sequence. Preferably, a transmembrane domain is able to crossthe plasma membrane and can, optionally, transduce an extracellularsignal into an intracellular response. Preferred transmembrane sequencesinclude, but are not limited to, sequences derived from CD8, ICAM-2,IL-8R, CD4, LFA-1, and the like.

Transmembrane sequences also can include GPI anchors, e.g., such as theDAF sequence (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT) (SEQ ID NO: 2)(see, e.g., Homans, et al., 1988, Nature 333(6170): 269-72; Moran, etal., 1991, J. Biol. Chem. 266: 1250); myristylation sequences (e.g.,such as the src sequence MGSSKSKPKDPSQR) (SEQ ID NO: 3) (see Cross, etal., 1984, Mol. Cell. Biol. 4(9): 1834; Spencer, et al., 1993, Science262: 1019-1024); and palmitoylation sequences (e.g., such as the GRK6sequence LLQRLFSRQDCCGNCSDSEEELPTR) (SEQ ID NO: 4).

Either the insertion sequence or the acceptor sequence can be alocalization sequence for localizing a molecule comprising the sequenceintracellularly. In one aspect, the localization sequence is a nuclearlocalization sequence. Generally, a nuclear localization sequence is ashort, basic sequence that serves to direct a polypeptide in which itoccurs to a cell's nucleus (Laskey, 1986, Ann. Rev. Cell Biol.2:367-390; Bonnerot, et al., 1987, Proc. Natl. Acad. Sci. USA 84:6795-6799; Galileo, et al., 1990, Proc. Natl. Acad. Sci. USA 87:458-462, 1990). Suitable nuclear localization sequences include, but arenot limited to, the SV40 (monkey virus) large T Antigen sequence(PKKKKKV) (SEQ ID NO: 5) (see, e.g., Kalderon, 1984, et al., Cell 39:499-509); the human retinoic acid receptor nuclear localization signal(ARRRRP) (SEQ ID NO: 6); NF κβ p50 sequence (EEVQRKRQKL) (SEQ ID NO: 7)(Ghosh et al., 1990, Cell 62: 1019); the NF κβ p65 sequence (EEKRKRTYE)(SEQ ID NO: 8) (Nolan et al., 1991, Cell 64: 961); and nucleoplasmin(Ala Val Lys Arg PAATLKKAGQAKKKKLD) (SEQ ID NO: 9) (Dingwall, et al.,1982, Cell 30:449-458).

The localization sequence can comprise a signaling sequence forinserting at least a portion of the fusion molecule into the cellmembrane. Suitable signal sequences include residues 1-26 of the IL-2receptor beta-chain (see, Hatakeyama et al., 1989, Science 244: 551; vonHeijne et al, 1988, Eur. J. Biochem. 174: 671); residues 1-27 of theinsulin receptor β chain (see, Hatakeyama, et al., 1989, supra);residues 1-32 of CD8 (Nakauchi, et al., 1985, PNAS USA 82: 5126) andresidues 1-21 of ICAM-2 (Staunton, et al., 1989, Nature (London) 339:61).

The localization sequence also can comprise a lysozomal targetingsequence, including, for example, a lysosomal degradation sequence suchas Lamp-2 (KFERQ) (SEQ ID NO: 10) (see, e.g., Dice, 1992, Ann. N.Y.Acad. Sci. 674: 58); a lysosomal membrane sequence from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI) (SEQ ID NO: 11) (see, e.g.,Uthayakumar, et al., 1995, Cell. Mol. Biol. Res. 41: 405) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF) (SEQ ID NO: 12) (see, e.g.,Konecki et al., 1994, Biochem. Biophys. Res. Comm. 205: 1-5).

Alternatively, the localization sequence can comprise a mitrochondriallocalization sequence, including, but not limited to: mitochondrialmatrix sequences, such as the MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ ID NO:13) of yeast alcohol dehydrogenase III (Schatz, 1987, Eur. J. Biochem.165:1-6); mitochondrial inner membrane sequences, such as theMLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO: 14) sequence of yeast cytochrome coxidase subunit IV (Schatz, 1987, supra); mitochondrial intermembranespace sequences, such as the MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLL YADSLTAEAMTA (SEQ ID NO:15) sequence of yeast cytochrome c1 (Schatz, 1987, supra); ormitochondrial outer membrane sequences, such as theMKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK (SEQ ID NO: 16) sequence ofyeast 70 kD outer membrane protein (see, e.g., Schatz, supra).

Other suitable localization sequences include endoplasmic reticulumlocalizing sequences, such as KDEL (SEQ ID NO: 17) from calreticulin(e.g., Pelham, 1992, Royal Society London Transactions B: 1-10) or theadenovirus E3/19K protein sequence LYLSRRSFIDEKKMP (SEQ ID NO: 18)(Jackson et al., 1990, EMBO J. 9: 3153); and peroxisome targetingsequences, such as the peroxisome matrix sequence (SKL) from Luciferase(Keller et al., 1987, Proc. Natl. Acad. Sci. USA 4: 3264).

In another aspect, the insertion sequence or acceptor sequence comprisesa secretory signal sequence capable of effecting the secretion of thefusion molecule from a cell (see, e.g., Silhavy, et al., 1985,Microbiol. Rev. 49: 398-418). This may be useful for generating a switchmolecule which can affect the activity of a cell other than a host cellin which it is expressed. Suitable secretory sequences, include, but arenot limited to the MYRMQLLSCIALSLALVTNS (SEQ ID NO: 19) sequence of IL-2(Villinger, et al., 1995, J. Immunol. 155: 3946); theMATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ ID NO: 20) sequence of growth hormone(Roskam et al., 1979, Nucleic Acids Res. 7: 30); theMALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO: 21) sequence of preproinsulin(Bell, et al., 1980, Nature 284: 26); the influenza HA protein sequence,MKAKLLVLLYAFVAGDQI (SEQ ID NO: 22) (Sekiwawa, et al., Proc. Natl. Acad.Sci. USA 80: 3563); or the signal leader sequence from the secretedcytokine IL4, MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO: 23).

In a further aspect, the insertion sequence or acceptor sequencecomprises a domain for binding a nucleic acid. The domain can comprise aDNA binding polypeptide or active fragment thereof from a prokaryote oreukaryote. For example, the domain can comprise a polypeptide sequencefrom a prokaryotic DNA binding protein such as gp 32; a domain from aviral protein, such as the papilloma virus E2 protein; or a domain froma eukaryotic protein, such as p53, Jun, Fos, GCN4, or GAL4. Novel DNAbinding proteins also can be generated by mutagenic techniques (see,e.g., as described in U.S. Pat. No. 5,198,346).

The insertion sequence or acceptor sequence also can comprise the Ca²⁺binding domain of a Ca²⁺ binding protein such as calmodulin,parvalbumin, troponin, annexin, and myosin or the ligand domain of abinding protein such as avidin, concanavalin A, ferritin, fibronectin,an immunoglobulin, a T cell receptor, an MHC Class I or Class IImolecule, a lipid binding protein, a metal binding protein, a chaperone,a G-protein coupled receptor, and the like.

In addition, the insertion or acceptor sequence can comprise thetransport domain of a transport protein such as hemerythrin, hemocyanin,hemoglobin, myoglobin, transferrin, lactoferrin, ovotransferrin, maltosebinding protein and transthyretrin.

In another aspect, the insertion or acceptor sequence can comprise theactive domain of a blood coagulation protein (e.g., a domain whichmediates blood clotting). Exemplary blood clotting proteins include, butare not limited to: decorsin, factor IX, factor X, kallikrein,plasmin/plasminogen, protein C, thrombin/prothrombin, and tissue-typeplasminogen activator.

In still another aspect, the insertion or acceptor sequence can comprisethe active domain of an electron transport protein (e.g., a domain whichconfers electron transport activity on a protein). Electron transportproteins include, but are not limited to, amicyanin, azurin, acytochrome protein, ferrodoxin, flavodoxin, glutaredoxin, methylaminedehydrogenase, plastocyanin, rubredoxin, and thioredoxin.

In a further aspect, the insertion sequence or acceptor sequencecomprises the catalytic and/or substrate binding site of an enzyme.Suitable enzymes from which such sites are selected include: aβ-lactamase; an acetylcholinesterase; an amylase; a barnase; adeaminase; a kinase (e.g., such as a tyrosine kinase or serine kinase);a phosphatase; an endonuclease; an exonuclease; an esterase; an enzymeinvolved in a metabolic pathway (e.g., fructose-1,6-bisphosphatase); aglycosidase; a heat shock protein; a lipase; a lysozyme; aneuramidase/sialidase; a phospholipase; a phosphorylase; apyrophosphatase; a ribonuclease; a thiolase; a polymerase; an isomerase(such as a mutase; triosephosphate isomerase, xylose isomerase,topoisomerase, gyrase); a lyase (such as aconitase, carbonic anhydrase,pyruvate decarboxylase); an oxidoreductase (such as alcoholdehydrogenase, aldose reductase, a catalase, cytochrome C, a peroxidase,a cytochrome p450, a dehydrogenase, a dihydrofolate reductase, aglyceraldehydes-3-phosphate dehydrogenase, a hydroxybenzoatehydroxylase, a lactate dehydrogenase, a peroxidase, a superoxidedismutase, a protease (such as actinidin, α-lytic protease,aminopeptidase, carboxypeptidase, chymosin, chymotrypsin, elastase,endopeptidase, endothiapepsin, HIV protease, Hannuka factor, papain,pepsin, rennin, substilisin, thermolysin, thermitase, and trypsin), atransferase (such as acetyltransferase, aminotransferase,carbamoyltransferase, dihyrolipoamide acetyltransferase, dihydrolipoyltransacetylase, dihydrolipoamide succinyltransferase, a nucleotidyltransferase, a DNA methyltransferase, a formyltransferase, aglycosyltransferase, a phosphotransferase, a phosphoribosyltransferase),a dehalogenase, a racemase, and the like.

The catalytic domain also can be a rhodanese homology domain such asforms the active site in various phosphatases and transferases (e.g.,such as found in the Cdc25 family of protein dual specificityphosphatases, the MKP1/PAC1 family of MAP-kinase phosphatases, thePyp1/Pyp2 family of MAP-kinase phosphatases, and certain ubiquitinhydrolases) (see, e.g., Hofmann, et al., 1998, J. Mol. Biol. 282:195-208).

Still other domains can include toxins such as cardiotoxin, conotoxin,erabutoxin, momorcharin, momordin, and ricin.

Other domains include, but are not limited to, signaling domains such asthe FHA domain, found in protein kinases and transcription factors suchas fork head, DUN1, RAD53, SPK1, cds1, MEK1, KAPP, NIPP1, Ki-67, fraH,and KIAA0170 (see, e.g., Hofmann and Bucher, 1995, Trends Biochem. Sci.20: 347-349); the death domain, a heterodimerization domain present inproteins involved in apoptotic signal transduction and the NFκβ pathway(such as TNFR1, FAS/APO1, NGFR, MORT1/FADD, TRADD, RIP, ankyrin, MyD88,unc-5, unc-44, DAP-kinase, Rb-binding p84, pelle, NFkB, and tubepolypeptides) (see, e.g., Hofmann and Tschopp, 1995, FEBS Lett. 371:321-323); and the G-protein desensitization domain (found in ARK1, GRK,G-protein coupled receptor kinases, eg1-10, GAIP, BL34 SST2, flbA, RGP3,RGP4Human G0/G1 switch regulatory protein 8, Human B-cell activationprotein BL34, and G-protein coupled receptor kinases) (see, e.g.,Hofmann and Bucher, “Conserved Sequence Domains in Cell Cycle RegulatoryProteins”, abstract presented at the joint ISREC/AACR meeting “Cancerand the Cell cycle”, January 1996 in Lausanne).

In one aspect, either the insertion or the acceptor sequence is alight-emitting polypeptide domain such as one obtained from a GreenFluorescent Protein, or modified, or mutant form thereof (collectivelyreferred to as a “GFP”). The wild-type GFP is 238 amino acids in length(Prasher, et al., 1992, Gene 111(2): 229-233; Cody et al., Biochem.32(5):1212-1218 (1993); Ormo, et al, 1996, Science 273: 1392-1395; andYang, et al., 1996, Nat. Biotech. 14: 1246-1251). Modified forms aredescribed in WO 98/06737 and U.S. Pat. No. 5,777,079. GFP deletionmutants also can be made. For example, at the N-terminus, it is knownthat only the first amino acid of the protein may be deleted withoutloss of fluorescence, while at the C-terminus, up to 7 residues can bedeleted without loss of fluorescence (see, e.g., Phillips, et al., 1997,Current Opin. Structural Biol. 7: 821).

The insertion sequence or acceptor sequence additionally can comprisethe light-reactive portion of a photoreceptor such asbacteriochlorophyll-A, bacteriorhodopsin, photoactive yellow protein,phycocyanin, and rhodopsin.

Additional domain sequences include ligand-binding domains ofligand-binding proteins. Such proteins include, but not limited to:biotin-binding proteins, lipid-binding proteins, periplasmic bindingproteins (e.g., maltose binding protein), lectins, serum albumins,immunoglobulins, T cell receptors, inactivated enzymes,pheromone-binding proteins, odorant-binding proteins,immunosuppressant-binding proteins (e.g., immunophilins such ascyclophilins and FK506-binding proteins), phosphate-binding proteins,sulfate-binding proteins, and the like. Additional binding proteins aredescribed in De Wolf and Brett, 2000, Pharmacological Reviews 52(2):207-236.]

The domain sequences of the proteins described above are known in theart and can be obtained from a database such as available at the NIHMolecular Modeling Homepage.

Additional Sequences in Fusion Proteins

Fusion molecules can further comprise domain sequences, as describedabove, in addition to insertion and acceptor sequences. Such domains cancomprise states which may or may not be coupled with the states of theother portions of the fusion molecule.

Additional sequences also can be included as part of the fusion moleculewhich do not alter substantially the states of the insertion sequence oracceptor sequence portion of the fusion molecule. For example, affinitytag sequences can be provided to facilitate the purification orisolation of the fusion molecule. Thus, His6 tags (SEQ ID NO: 99) can beemployed (for use with nickel-based affinity columns), as well asepitope tags (e.g., for detection, immunoprecipitation, or FACSanalysis), such as myc, BSP biotinylation target sequences of thebacterial enzyme BirA, flu tags, lacZ, GST, and Strep tags I and II.Nucleic acids encoding such tag molecules are commercially available.

Stability sequences can be added to the fusion molecule to protect themolecule from degradation (e.g., by a protease). Suitable stabilitysequences include, but are not limited to, glycine moleculesincorporated after the initiation methionine (e.g., MG or MGG) toprotect the fusion molecule from ubiquitination; two prolinesincorporated at the C-terminus (conferring protection againstcarboxypeptidase action), and the like.

In some aspects, the fusion molecule can include a linking or tetheringsequence between insertion and acceptor sequences or between insertionor acceptor sequences and other domain sequences. For example, usefullinkers include glycine polymers, glycine-serine polymers,glycine-alanine polymers, alanine-serine polymers, alanine polymers, andother flexible linkers as are known in the art (see, e.g., Huston, etal., 1988, Proc. Natl. Acad. Sci. USA 85: 4879; U.S. Pat. No.5,091,513).

These additional sequences can be included to optimize the properties ofthe fusion molecules described herein.

Exemplary Fusion Molecules

Exemplary fusion molecules according to the invention are describedherein and illustrated schematically in FIGS. 5A-G. Methods of usingthese fusion molecules as molecular switches in cells are furtherdescribed infra. It should be apparent to those of skill in the art thatthese are merely examples of combinations of insertion and acceptorsequences that can be used to form a molecular switch, and are notintended to be limiting.

In one aspect, the invention provides a fusion protein comprising aninsertion sequence and an acceptor sequence, wherein either the insertedsequence or the acceptor sequence binds to a DNA molecule, and whereinDNA binding activity is coupled to the response of the respective othersequence of the fusion molecule to a signal. (FIG. 5A.)

In a further aspect, the fusion molecule comprises a molecular switchfor controlling a cellular pathway. The fusion molecule comprises aninsertion sequence and an acceptor sequence and the states of theinsertion sequence and acceptor sequence are coupled, such that thestate of either the insertion sequence or the acceptor sequencemodulates the activity or expression of a molecular pathway molecule ina cell. Preferably, modulation of activity or expression occurs when therespective other portion of the fusion molecule responds to a signal,e.g., binds to an exogenous or endogenous binding molecule (e.g.,ligands, small molecules, ions, metabolites, and the like), responds toelectrical or chemical properties of a cell, or responds to the opticalenvironment in which a cell is found (e.g., responding to the presenceor absence of a particular wavelength(s) of light) (FIG. 5B).

The fusion molecule also can comprise an insertion sequence and acceptorsequence, wherein either the inserted sequence or the acceptor sequenceassociates with a bio-effective molecule, and disassociates from thebio-effective molecule, when the respective other sequence of the fusionbinds to a cellular marker of a pathological condition (FIG. 5C). Suchmarkers can comprise polypeptides, nucleic acids, glycoproteins, lipids,carbohydrates, small molecules, metabolites, pH, ions and the like.Examples of cellular markers of pathological conditions include, but arenot limited to cancer-specific or tumor-specific antigens,pathogen-encoded polypeptides (e.g., viral-, bacterial-, protist-, andparasite-encoded polypeptides) as are known in the art.

In another aspect, the insertion sequence or the acceptor sequencelocalizes the fusion molecule intracellularly. Preferably, intracellularlocalization is coupled to the binding of the fusion molecule to abio-effective molecule (FIG. 5D).

In still another aspect, the fusion molecule is capable of switchingfrom a non-toxic state to a toxic state. Either the insertion sequenceor acceptor sequence may bind to a cellular marker of a pathology (e.g.,such as a tumor antigen). Binding of the marker to the fusion proteinswitches the fusion protein from a non-toxic state or a less toxic stateto a toxic state. Similarly, a marker of a healthy cell could be used asa trigger to switch a fusion molecule from a toxic state to a non-toxicstate, or to a less toxic state (FIG. 5E).

In yet a further aspect, the fusion molecule can affect a metabolicstate in a cell. Either the insertion sequence or the acceptor sequencemay bind to an effector molecule. Binding of the effector molecule tothe fusion protein triggers enzymatic activity by the enzyme. (See FIG.5F and Examples, infra.)

The invention also provides a sensor molecule comprising an insertionsequence and an acceptor sequence, wherein either the insertion sequenceor the acceptor sequence binds to a target molecule and wherein therespective other sequence generates a signal in response to binding(FIG. 5G).

Methods of Using Molecular Switches

In one aspect, the invention provides a method for using a molecularswitch to modulate a cellular activity. The cellular activity caninclude an enzyme activity, the activity of one or more cellular pathwaymolecules, the transduction of a signal, and the like. Modulation maydirect, e.g., the switch itself may alter the activity, or indirect,e.g., the switch may function by delivering a bio-effective molecule tothe cell which itself modulates the activity. Modulation can occur invitro (e.g., in cell culture or in a cell extract) or in vivo (e.g.,such as in a transgenic organism). Molecular switches comprising fusionpolypeptides also can be administered to a cell by delivering suchmolecules systemically (e.g., through intravenous, intramuscular, orintraperitoneal injections, or through oral administration of either thepolypeptides themselves or nucleic acids encoding the polypeptides) orlocally (e.g., via injection into a tumor or into an open surgicalfield, or through a catheter or other medical access device, or viatopical administration).

In one aspect, molecular switches are used to conditionally modulate anenzymatic activity in a cell. For example, a switch molecule can beintroduced into a cell that comprises an insertion sequence or acceptorsequence which provides the enzymatic activity. Catalysis by theinsertion or acceptor sequence is coupled to the response of therespective other portion of the fusion molecule to a signal, such asbinding of the other portion to a molecule (e.g., such as an agentadministered to the cell or a naturally occurring small molecule),exposure of the cell to particular chemical conditions (e.g., such aspH), electrical conditions (e.g., potential differences), opticalconditions (e.g., exposure of the cell to light of specificwavelengths), magnetic conditions and the like.

In another aspect, a molecular switch is provided which modulates theactivity or expression of a molecular pathway molecule in a cell. FIG.5B shows an example of a switch molecule comprising a pathway moleculewhich is conditionally active in the presence of a signal (schematicallyillustrated as “ ” in the Figure). The switch molecule is used to altera cell signaling pathway, e.g., altering the expression and/or activityof downstream pathway molecules (turning such molecules ON or OFF, oraltering the level of expression and/or activity of such molecules). Indoing so, the switch molecule can be used to regulate the fate of one ormore cells.

Similarly, the molecular switches according to the invention can be usedto control metabolic pathways, e.g., providing a fusion molecule whichprovides an enzymatic activity coupled to the binding of a smallmolecule, or response to some other signal (as shown in FIG. 5F).Preferably, modulation of the enzyme activity in response to the signal,in turn, modulates the expression and/or activity of moleculesdownstream in the metabolic pathway.

More preferably, the states of the fusion molecules are coupled to asignal, such as the presence of an exogenous or endogenous bindingmolecules to which either the insertion sequence or acceptor sequencebinds. The ability of the fusion molecule to control a pathway can bemonitored by examining the expression and/or activity of pathwaymolecules which act downstream of a pathway molecule whose expressionand/or activity is being modulated/controlled by the fusion molecule.Preferably, control of the pathway is coupled to the presence of thesignal, e.g., binding of the fusion molecule to the exogenous orendogenous binding molecule, the presence of particular electrical orchemical properties of a cell, the presence or absence of particularwavelength(s) of light, and the like.

Pathways of interest include the phosphatidylinositol-specificphospholipase pathway, which is normally involved with hydrolysis ofphosphatidylinositol-4,5-bisphosphate and which results in production ofthe secondary messengers inositol-1,4,5-trisphosphate anddiacylglycerol. Other pathways include, but are not limited to: a kinasepathway, a pathway involving a G protein coupled receptor, aglucerebrosidase-mediated pathway, a cylin pathway, an anaerobic oraerobic metabolic pathway, a blood clotting pathway, and the like.

In still another aspect, a fusion molecule is provided which delivers abio-effective molecule (e.g., a drug, therapeutic agent, diagnostic orimaging agent, and the like) to a cell. In one scenario, shown in FIG.5C, the fusion molecule comprises an insertion or acceptor sequencewhich binds to the bio-effective molecule, while the respective otherportion of the fusion binds to a cellular marker that is a signature ofa pathology, e.g., a small molecule, polypeptide, nucleic acid,metabolite, whose expression (presence or level) is associated with thepathology. Preferably, the fusion molecule releases the bio-effectivemolecule only in the presence of the marker of the pathology.

FIG. 5D shows an alternative method of transporting a bio-effectivemolecule. In this aspect, the insertion sequence or acceptor sequencecomprises a transport sequence for transporting a bio-effective moleculebound to the fusion molecule intracellularly. Preferably, the insertionsequence and acceptor sequence are functionally coupled such that aconformational change in the transport sequence is coupled tointracellular release of the bio-effective agent. Successful deliverycan be monitored by measuring the effect of the bio-effective agent(e.g., its ability to mediate a drug action or therapeutic effect, or toimage a cell). More preferably, the conformation change occurs uponresponse of the respective other portion of the fusion to a signal(indicated schematically in the Figure as “ ”), enabling conditionalintracellular transport of the bio-effective molecule. When thebio-effective agent is delivered to one or more cells in an organism,the effect of the agent on the physiological responses of the organismcan be monitored, e.g., by observing clinical or therapeutic endpointsas is routine in the art. Where the bio-effective molecule is an imagingmolecule, the localization of the bio-effective molecule in the organismcan be monitored by MRI, X-ray, angiography, and the like.

In still another aspect, the invention provides a method for killingundesired cells, such as abnormally proliferating cells, e.g., cancercells (FIG. 5E). For example, a fusion protein comprising aconditionally toxic molecule which targets to a cell having a pathologycan be administered to a cell (or an organism comprising the cell).Preferably, the toxic state of the fusion protein is coupled to theresponse of the fusion protein to a signal, such as exposure to a markerof a pathology, causing the fusion protein to switch from a non-toxicstate to a toxic state when it encounters the cell comprising thepathology. In one aspect, the change in state from a toxic to anon-toxic or less toxic molecule is coupled to binding of the fusionprotein to the marker of the pathology.

In a further aspect, a fusion molecule is provided for regulating anactivity of a nucleic acid regulatory sequence in vitro or in vivo.Activities which can be regulated include transcription, translation,replication, recombination, supercoiling, and the like (FIG. 5A).Preferably, fusion molecules are selected in which binding of theinsertion sequence or acceptor sequence of the fusion molecule to thenucleic acid regulatory sequence is coupled to the response of therespective other sequence of the fusion molecule to a signal. Suchfusion molecules can be used to create cells with conditional knockoutsor knock-ins of a gene product whose expression is mediated by theactivity of the nucleic acid regulatory sequence to which the fusionmolecule binds, e.g., by providing or withdrawing the signal asappropriate. In one aspect, the signal is a drug or therapeutic agent.In another aspect, the signal is a change in pH, a change in cellularpotential, or a change in exposure of a cell (and/or organism) to light.For example, a probe for delivering particular wavelengths of light canbe used to provide a highly localized signal to a cell expressing afusion molecule in vivo.

In still a further aspect, the fusion molecules according to theinvention comprise sensor molecules that can be used to detect targetanalytes in vitro or in vivo (FIG. 5G). Target analytes include, but arenot limited to: small molecules, metabolites, lipids, glycoproteins,carbohydrates, amino acids, peptides, polypeptides, proteins, antigens,nucleotides, nucleic acids, cells, cell organelles, and small organisms(e.g., microorganisms such as bacteria, yeast, protests, and the like).

The fusion molecule can be exposed to a target molecule in solution orstably associated with a solid support that can be exposed to a samplesuspected of containing the target molecule. Alternatively, the fusionmolecule can be expressed in a cell, i.e., for detecting intracellularor extracellular targets (for example, where the fusion moleculecomprises an extracellular binding domain). Analyte present in thesample will bind to the fusion molecule, triggering production of asignal by the signaling portion of the molecule. Suitable signalingmolecules from which this portion can be obtained include moleculescapable of emitting light, e.g., such as GFP, or modified, or mutantforms thereof (e.g., EGFP, YFP, CFP, EYFP, ECFP, BFP, and the like).Other signaling molecules include electron transferring domains (e.g.,such that the electrical characteristics of the fusion molecule can bemonitored to provide a measure of target analyte), binding domains(e.g., domains capable of binding to a labeled molecule), and catalyticdomains (e.g., β-lactamase, luciferase, alkaline phosphatase, and thelike).

Signaling molecules which comprise catalytic domains can be detected bymonitoring changes in the level of a fluorescent substrate. For example,when the catalytic domain is obtained from β-lactamase, fluorescentsubstrates such as CCF2/FA and CCF2/AM can be used (see, e.g.,Zlokarnik, et al., Science 279: 84-88 (1998)).

In a further aspect, the invention provides a method for modulating acellular response by conditionally providing a pair of fusionpolypeptides to a cell to mediate the response. For example, the pair offusion polypeptides can comprise a binding activity, an enzymaticactivity, a signaling activity, a metabolic activity, and the like. Inone aspect, the pair of fusion polypeptides modulate transcription,translation, or replication of the cell and/or alters a cellularphenotype in response to a signal

Host Cells for Expressing Fusion Molecules

Fusion molecules according to the invention can be expressed in avariety of host cells, including, but not limited to: prokaryotic cells(e.g., E. coli, Staphylococcus sp., Bacillus sp.); yeast cells (e.g.,Saccharomyces sp.); insect cells; nematode cells; plant cells; amphibiancells (e.g., Xenopus); fish cells (e.g., zebrafish cells); avian cells;and mammalian cells (e.g., human cells, mouse cells, mammalian celllines, primary cultured mammalian cells, such as from dissectedtissues).

The molecules can be expressed in host cells isolated from an organism,host cells which are part of an organism, or host cells which areintroduced into an organism. In one aspect, fusion molecules areexpressed in host cells in vitro, e.g., in culture. In another aspect,fusion molecules are expressed in a transgenic organism (e.g., atransgenic mouse, rat, rabbit, pig, primate, etc.) that comprisessomatic and/or germline cells comprising nucleic acids encoding thefusion molecules.

Fusion molecule also can be introduced into cells in vitro, and thecells (e.g., such as stem cells, hematopoietic cells, lymphocytes, andthe like) can be introduced into the host organism. The cells may beheterologous or autologous with respect to the host organism. Forexample, cells can be obtained from the host organism, fusion moleculesintroduced into the cells in vitro, and then reintroduced into the hostorganism.

EXAMPLES

The invention will now be further illustrated with reference to thefollowing examples. It will be appreciated that what follows is by wayof example only and that modifications to detail may be made while stillfalling within the scope of the invention.

Example 1 Generating Fusion Molecules by Circular Permutation and DomainInsertion

This example describes a model system combining E. coli maltose bindingprotein (“MBP”) as the acceptor polypeptide sequence and thepenicillin-hydrolyzing enzyme TEM1 β-lactamase (“BLA”) as the insertionpolypeptide sequence. The BLA-MBP fusion molecule was chosen todemonstrate the circular permutation domain insertion strategy forproducing molecular switches capable of coupling the functions of thetwo proteins. The desired property of the model switch is the ability tomodulate β-lactamase activity through changes in maltose concentration.FIG. 1 is a schematic summary diagram of the cloning steps used in thisExample.

Linkers for Circular Permutation

In order to circularly permute a gene it is generally necessary toinclude DNA that codes for a linker to link the original N- andC-termini. We chose to test two different linkers. For the first (the“DKS linker”), β-lactamase was randomly circularly permuted by fusingthe 5′- and 3′-ends with a DNA sequence coding for the tripeptide linkerDKS, previously found in a combinatorial library of linkers to be mostconducive for circularly permuting β-lactamase when the new N- andC-termini were located at a specific location (Osuna, Pérez-Blancas etal. 2002). For the second selected linker, (the “GSGGG linker”) (SEQ IDNO: 1), the β-lactamase was randomly circularly permuted by fusing the5′- and 3′-ends with a DNA sequence coding for the flexible pentapeptidelinker GSGGG (SEQ ID NO:1)

Preparation of BLA Insert DNA

The β-lactamase gene fragment bla [24-286] (encoding amino acids 24-286)was selected for this study. DNA coding for amino acids 1-23 was notdesired because it codes for the signal sequence that targetsβ-lactamase to the periplasm and is not part of the mature, activeβ-lactamase. The fragment was amplified by PCR from pBR322 such that itwas flanked by EarI or BamHI restriction enzyme site sequences codingfor the linkers described above and cloned into pGem T-vector (Promega)to create pBLA-CP(DKS) (FIG. 2) and pBLA-CP(GSGGG) (“GSGGG” disclosed asSEQ ID NO: 1), (FIG. 3).

One hundred and thirty micrograms of pBLA-CP(GSGGG) (“GSGGG” disclosedas SEQ ID NO: 1) was digested with 2000 units of BamHI and 140micrograms of pBLA-CP(DKS) was digested with 600 units of EarI in thebuffers and conditions recommended by the manufacturer of therestriction enzyme. The fragment containing the BLA gene was purified byagarose gel electrophoresis using the QIAquick™ gel purification kit.This DNA was treated with T4 DNA ligase under dilute concentrations tocyclize the DNA (18 hours at 16° C. with 600 Weiss units of T4 DNAligase in the presence of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM ATP, 25 ug/ml BSA in a total volume of 5.1 ml). Theligation reaction was stopped by incubation at 65° C. for 20 minutes.The DNA was concentrated by vacufuge and desalted using the QIAquick™PCR purification kit. Circular fragments were purified by agarose gelelectrophoresis using the QIAquick™ gel purification kit.

The conditions for DNaseI digestion were determined experimentally byadding different amounts of DNaseI and analyzing the digested productsby agarose gel electrophoresis. The digestion conditions were chosensuch that a significant fraction of DNA was undigested in order maximizethe amount of linear DNA that only had one double strand break. Ingeneral, approximately 1 milliunit of DNaseI per microgram of DNA (at aconcentration 10 micrograms/ml) for an 8 minute digestion at 22° C. wasfound to be optimal. Sometimes more or less DNaseI was required and thuspreferably for each library constructed the correct amount of DNaseI isdetermined experimentally by test digestions. The following conditionsare a representative example. Six micrograms of circular DNA wasdigested with 6 milliunits of DNase I (Roche) for 8 minutes at 22° C. inthe presence of 50 mM TrisHCl (pH 7.4), 1 mM MnCl₂ and 50 micrograms/mlBSA in 0.6 ml reaction volume. The reaction was stopped by adding EDTAto a concentration of 5 mM. The DNA was desalted using the QIAquick™ PCRpurification kit and repaired by 6 units of T4 DNA polymerase and 6Weiss Units of T4 DNA ligase at 12° C. for 15 minutes in the presence of100 micromolar dNTP, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, mMdithiothreitol, 1 mM ATP and 25 ug/ml BSA. The repaired, linear DNA waspurified by agarose gel electrophoresis using the QIAquick gelpurification kit. This circularly permuted DNA was in a form ready forinsertion into another plasmid.

Preparation of Target DNA for Random Domain Insertion Libraries

Forty μg of pDIM-C8-Mal was digested with DNaseI (0.01 units) for 8minutes at 22° C. in the presence of 50 mM Tris-HCl, pH 7.4, 10 mM MnCl₂and 50 μg/ml BSA in a total volume of 1 ml. The reaction was quenched bythe addition of EDTA to a concentration of 5 mM and the solution wasdesalted using four Qiaquick™ PCR purification columns into 200 μAelution buffer which was subsequently concentrated by vacufuge. Nicksand gaps were repaired by incubating at 12° C. for 1 hour in a totalvolume of 120 μl in the presence of T4 DNA polymerase (15 units) and T4DNA ligase (12 Weiss units) in the presence of 50 mM Tris-HCl, pH 7.5,10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA and 125 μM dNTPs. Thereaction was stopped by incubating at 80° C. for 10 minutes. Sodiumchloride was added to 100 mM and the DNA was dephosphorylated by addingalkaline phosphatase (60 units) and incubating at 37° C. for 1 hour. TheDNA was desalted as before and the linear DNA (corresponding to therandomly linearized pDIM-C8-Mal) was isolated from circular forms of theplasmid by agarose gel electrophoresis using the Qiaquick gelpurification kit.

Preparation of Target DNA for Site-Specific Insertion Libraries

Referring to FIG. 4, plasmid pDIM-C8-Mal was modified using overlapextension (Horton, Hunt et al. 1989) to be suitable for insertion of thecircularly permuted BLA at two specific sites: (a) between MBP [1-165]and MBP [164-370] and (b) at the C-terminus of MBP. The plasmids weremodified in analogous ways. The modifications for insertion between MBP[1-165] and MBP [164-370] to create plasmid pDIMC8-MBP(164-165) aredescribed below and shown in FIG. 4. Two inverted SapI sites wereinserted between DNA coding for MBP [1-165] and MBP [164-370] in such amanner that digestion with SapI and subsequent filling in of theresulting overhangs using Klenow polymerase in the presence of dNTPsresults in a perfectly blunt MBP [1-165] on one side and a perfectlyblunt MBP [164-370] on the other side. This is achieved by virtue of thefact that SapI is a type IIS restriction enzyme that cuts outside of itsrecognition sequence. Other type IIS restriction enzymes could have beenused. Non-type IIS restriction enzymes could also be used if it isacceptable to have their recognition site as part of the gene fragmentthat is being inserted into.

Three micrograms of pDIMC8-MBP(164-165) was digested with 6 units ofSapI at 37° C. in the presence of 50 mM potassium acetate, 20 mMTris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9), 100ug/ml BSA for 2.5 hours. The DNA was desalted using the QIAquick™ PCRpurification kit and repaired with 5 units of Klenow at 25° C. for 20minutes in the presence of 33 micromolar dNTPs, 100 mM NaCl, 50 mMTris-HCl, 10 mM MgCl₂ and 1 mM dithiothreitol (pH 7.9). The enzyme washeat inactivated by incubation at 75° C. for 20 minutes. Sodium chloridewas added to 100 mM and ten units of Calf Intestinal Phosphatase wasadded and the solution was incubated for 1 hour at 37° C.Dephosphorylation was performed to prevent recircularization of thevector without receiving an insert in the subsequent ligation step. Thevector DNA was purified by agarose gel electrophoresis using theQIAquick™ gel purification kit.

Ligation of Inserts into Target DNA

Insert DNA (85 ng) comprising the circularly permuted BLA was ligated tothe prepared target DNA (100 ng) at 22° C. overnight in the presence ofT4 DNA ligase (30 Weiss units) and the ligase buffer provided by themanufacturer in a total volume of 13 μl. After ethanol precipitation,10% of the ligase-treated DNA was electroporated into 50 μl Electromax™DH5α-E electrocompetent cells (Invitrogen, Carlsbad, Calif.).Transformed cells were plated on large (248 mm×248 mm) LB agar platesupplemented with 50 μg/ml chloramphenicol (Cm). The naïve domaininsertion library was recovered from the large plate (Ostermeier, Nixonet al. 1999) and stored in frozen aliquots.

Screening for Allosteric Enzymes

The libraries were diluted from frozen aliquots and plated on LB platescontaining different concentrations of ampicillin (Tables 1 and 2). Anumber of colonies were picked (Tables 3) and grown in LB overnight in96 well plates (0.5 ml/well) in the presence of 1 mM IPTG and 50 μg/mlCm.

TABLE 1 Library Statistics. Table discloses “GSGGG” as SEQ ID NO: 1.Number of Number Increase in Library library of unique velocity (ofsize (Number members Number of switches nitrocefin of that can coloniesfound hydrolysis in transformants grow on 50 screened for with ≧2-presence of Insertion site Linker in with BLA ug/m1 AMP switching foldmaltose) of in MBP BLA insert). (see Table 2) (see Table 3) effect*best switch 164-165 DKS 0.44 × 10⁶ 515 848 2   +97% GSGGG 1.05 × 10⁶ 3611248 1  −250% C-terminus DKS 1.03 × 10⁶ 2414 576 0 GSGGG 0.30 × 10⁶ 16151920 1-4  +234% random DKS 0.41 × 10⁶ 191 384 0 GSGGG 1.20 × 10⁶ 11563312 5 +1650% *≧2-fold change in velocity of nitrocefin hydrolysis inthe presence of 5 mM maltose.

TABLE 2 Number of Library Members Capable of Grow onPlates with Ampicillin (With or Without Maltose).Table discloses “GSGGG” as SEQ ID NO: 1. Ampi- T164- T164- cillinMaltose? 165 165 EE EE Random Random (μg/ml) (5 mM) DKS GSGGG DKS GSGGGDKS GSGGG 5 no 734 878 7052 3510 nd 2458 50 no 394 294 1747 1159 nd 783200 no 220 nd 1080 298 nd nd 1000 no nd 74 nd nd nd 60 5 yes 1098 7618354 4056 nd 1969 50 yes 515 361 2414 1615 191 1156 200 yes 182 240 1525630 nd 272 1000 yes nd 88 nd nd nd 34

TABLE 3 Number of Library Members Screened (Picked from Plates with Indicated Ampicillin and Maltose Levels). Table discloses “GSGGG”as SEQ ID NO: 1. Ampicillin Maltose? T164-165 T164-165 EE EE RandomRandom (μg/ml) (5 mM) DKS GSGGG DKS GSGGG DKS GSGGG 5 no — 96 — 288 — 9650 no — — — — — — 200 no — — — — 480 — 1000 no — — — — — — 5 yes 96 192864 — 768 — 50 yes 672 576 576 768 384 960 200 yes 80 384 — — — 10081000 yes — — — — — — EE = end-to-end (insertion at C-terminus)

Next, 50 μl of PopCulture (Novagen) and 2.5 unit of benzonase nucleasewas added to each well and incubated for 15 minutes at room temperatureto lyse the cells. The cells debris and any unlysed cells were pelletedby centrifugation and supernatant was recovered. In 96-well format, 60μA of lysate was assayed for hydrolysis of nitrocefin (50 μM) bymonitoring the increase in absorbance at 490 nm in 100 mM sodiumphosphate buffer, pH 7.0, both with and without 5 mM maltose. Any lysatein which there was a difference in rate of more than 2-fold (betweenwith and without maltose) was selected for retesting and furtherinvestigation.

Confirmation and Identification of Positives

Library members identified as having more than 200% switching activityin the 96-well plate screen were grown 24-48 hours in 100 ml LB media in500 ml shaker flasks at 22° C. without IPTG. The cells were pelleted andresuspended in 8 ml assay buffer (100 mM sodium phosphate buffer, pH7.0) and lysed by French press. The soluble fraction of this lysate wasassayed for hydrolysis of nitrocefin (50 μM) at 22° C. as previouslydescribed (Guntas and Ostermeier 2004) both with and without 5 mMmaltose. Initial rates were determined from absorbance at 486 nmmonitored as a function of time. The enzyme was incubated at the assaytemperature in the absence or presence of 5 mM maltose for four minutesprior to performing the assay. All assays contained 100 mM sodiumphosphate buffer, pH 7.0. Library members for which there was adifference in the initial rate of more than about 2-fold were sequenced(Table 4). Switches RG-5-169 and RG-200-13 were also assayed in thepresence of 5 mM sucrose or 5 mM glucose. Neither sugar affected thevelocity of nitrocefin hydrolysis, indicating that the switching effectwas specific for maltose, a ligand to which MBP binds.

TABLE 4 Switching Effect of Selected BLA-MBP Molecular Switches. Tablediscloses “GSGGG” as SEQ ID NO: 1. Switching Switch Sequence effect*IFG-5-277 MBP[1-165]-BLA[218-286]-GSGGG- −250% BLA[24-215]-MBP[164-370]IFD-5-7 MBP[1-165]-BLA[110-286]-DKS- +96% BLA[24-107]-MBP[164-370]IFD-5-15 MBP[1-165]-BLA[168-286]-DKS- +97% BLA[24-170]-MBP[164-370]EEG-50-530 MBP[1-370]-BLA[114-286]-GSGGG- +228% BLA[24-112]-GSQQHEEG-50-251 MBP[1-370]-BLA[114-286]-GSGGG- +234% BLA[24-114]-K RG-5-169MBP[1-338]-BLA[34-286]-GSGGG- +855% BLA[24-29]-MBP[337-370] RG-200-13MBP[1-316]-BLA[227-286]-GSGGG- +1650% BLA[24-226]-S-MBP[319-370]*Percent change in velocity of nitrocefin hydrolysis (50 μM nitrocefin)in the presence of 5 mM maltose in 100 mM sodium phosphate buffer, pH7.0.

Analysis of Purified Switch RG-200-13

A 6×His tag (SEQ ID NO: 99) was added to the C-terminus of RG-200-13(also termed “RG13” in Examples below) and the fusion was purified aspreviously described (Guntas and Ostermeier 2004). The protein waspurified to approximately 60% purity. The kinetic constants and bindingconstants were determined from Eadie-Hofstee plots and Eadie plotequivalents, respectively, using a spectrophotometric assay fornitrocefin hydrolysis. Initial rates for nitrocefin hydrolysis weredetermined from absorbance at 486 nm monitored as a function of time.The enzyme was incubated at the assay temperature in the absence orpresence of saccharide for four minutes prior to performing the assay.All assays contained 100 mM sodium phosphate buffer, pH 7.0. Thedissociation constant for maltose was determined using change invelocity of nitrocefin hydrolysis as a signal.

Only sugars known to bind to MBP had an effect on nitrocefin hydrolysis(Table 5). Those sugars that produce a large conformational change uponbinding MBP (Quiocho, Spurlino et al. 1997) (maltose and maltotriose)produced the largest change in the velocity of nitrocefin hydrolysis.Beta-cyclodextrin, which produces a small conformational change uponbinding MBP (Evenas, Tugarinov et al. 2001), has a small effect. Theeffect of maltotetraitol is intermediate, consistent with the fact thatmaltotetraitol-binding to MBP results in a mixture of open and closedstructures (Duan, Hall et al. 2001).

TABLE 5 Sugar Dependence of Switching Effect of RG-200-13*. Change invelocity of nitrocefin hydrolysis in Sugar Binds to MBP? presence ofsugar Sucrose No −5% Lactose No −4% Galactose No −3% Maltose Yes +1800%Maltotriose Yes +1700% Maltotetraitol Yes +400% β-cyclodextrin Yes +150%*50 μM nitrocefin, 100 mM sodium phosphate buffer, pH 7.0, 22° C., 5 mMsugar except for β-cyclodextrin (3 mM).

The kinetic parameters of RG-200-13 are reported in Table 6. The kineticparameters of RG-200-13 at 22° C. in the presence of maltose(k_(cat)=˜520 s⁻¹; K_(m)=˜85 M) are very similar to previously reportedvalues for TEM-1 β-lactamase at 30° C. (k_(cat)=930 s⁻¹; K_(m)=52 μM)(Raquet, Lamotte-Brasseur et al. 1994) indicating that RG-200-13 isessentially a fully functional TEM-1 β-lactamase in the presence ofmaltose. The k_(cat)/K_(m) in the presence of 5 mM maltose isapproximately 25-fold higher than in the absence of maltose. The K_(d)for maltose binding to RG-200-13 at 22° C. was ˜5 μM, similar to theK_(d) previously reported for maltose binding to MBP (1-1.5 μM)(Schwartz, Kellermann et al. 1976).

TABLE 6 Kinetic Parameters of Nitrocefin Hydrolysis of RG-200-13Molecular Switch. k_(cat) (s⁻¹) K_(m) (μM) No No mal- 5 mM mal- 5 mMk_(cat)/K_(m) Substrate tose maltose Ratio^(a) tose maltose Ratio^(a)Ratio^(a) nitrocefin ~80 ~520 ~6.5 ~325 ~85 ~0.26 ~25 ^(a)(withmaltose)/(without maltose). Conditions: 100 mM sodium phosphate buffer,pH 7.0, 22° C.

The effect of 5 mM maltose on other substrates of BLA is shown in Table7. Maltose binding had the largest effect on cephalothin (of thesubstrates tested), with the velocity of cephalothin hydrolysis being32-fold higher in the presence of maltose than in its absence. Based onthe effects on other substrates, the actual switching effect onk_(cat)/K_(m) for cephalothin is likely to be much higher than 32-fold.

TABLE 7 Effect of Maltose on Other Substrates of Switch RG-200-13.Approximate fold increase Substrate K_(m) for in velocity of nitrocefinconcen- TEM-1 β- hydrolysis in the presence Substrate trationlactamase^(a) of 5 mM maltose cephalothin 250 μM 246 μM  32 ampicillin100 μM 32 μM 26 500 μM 10 benzylpenicillin 100 μM 19 μM 17 500 μM 7carbenicillin  1 mM ? 4 oxacillin  1 mM  3 μM 5 Conditions: 100 mMsodium phosphate buffer, pH 7.0, 22° C. ^(a)(Raquet, Lamotte-Brasseur etal. 1994)

The fact that the magnitude of the switching effect of RG-200-13 isdependent on substrate identity and concentration strongly argues thatmaltose is converting the protein from a less active to a more activeconformation. If an alternative explanation, i.e., that maltose affectsthe equilibrium between unfolded (inactive) and folded (active) forms ofthe protein were true, the observed switching effect would beindependent of the substrate being tested and independent of substrateconcentration, which was not the case.

Example 2 Construction and Characterization of a Molecular SwitchesCreated by In Vitro Recombination of Non-Homologous Genes

This example describes further studies of exemplary molecular switchescomprising BLA-MBP fusions made by the methods of the invention.

Materials and Methods

All restriction enzymes, T4 DNA ligase, T4 DNA polymerase, and calfintestinal phosphatase were purchased from New England Biolabs (Beverly,Mass.). pGEM T-vector cloning kit and Taq polymerase were purchased fromPromega (Madison, Wis.). DNAseI was purchased from Roche Biochemicals(Indianapolis, Ind.). Qiaquick™ PCR purification kit and Qiaquick gelextraction kit were purchased from Qiagen (Valencia, Calif.). Popculturereagent, rLysozyme, benzonase nuclease, and His-tag protein purificationkit were purchased from Novagen (Madison, Wis.). Oligonucleotides andElectromax™ DH5α-E electrocompetent cells were purchased from Invitrogen(Carlsbad, Calif.). Nitrocefin was purchased from Oxoid (Hampshire, UK).Maltotriose and β-cyclodextrin were purchased from Sigma (St. Louis,Mo.). Antibiotics, maltose, lactose, galactose and sucrose werepurchased from Fisher Scientific (Pittsburgh, Pa.).

Random Circular Permutation

The portion of the bla gene encoding the mature BLA was fused to asequence coding for a GSGGG linker (SEQ ID NO: 1) and containing a BamHIsite by PCR amplification using the forward primer:

(SEQ ID NO: 24) 5′-TGCC GGATCCGGCGGTGGCCACCCAGAAACGCTGGTG-3′and the reverse primer (SEQ ID NO: 25) 5′-GTCTGA GGATCCCCAATGCTTAATCAGTGA-3′.

Portions of the primers encoding the GSGGG linker (SEQ ID NO: 1) areunderlined and the BamHI site is highlighted in bold. The PCR productwas desalted using Qiaquick PCR purification kit and ligated to the pGEMT-vector to create plasmid pGEMT-BLA. One hundred and fifty μg ofpGEMT-BLA was digested with 1000 units of BamHI and the DNA fragmentthat encodes BLA was gel purified using Qiaquick gel purification kit.Eighteen μg of this DNA was cyclized by ligation at 16° C. for 18 hoursin a reaction volume of 5.1 ml in the presence of ligase buffer (50 mMTris-HCl, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA pH 7.5) and 600Weiss units of T4 DNA ligase. After heat inactivation of the ligase, theconcentrated reaction mixture was desalted and the circular DNA waspurified by agarose gel electrophoresis using Qiaquick Gel Extractionkit.

To introduce the random double stranded break, 8 μg of circular DNA wasdigested with 8 milliunits of DNAse I in the presence of 50 mM Tris-HCl,pH 7.4, 10 mM MnCl₂ and 50 μg/ml BSA in a total volume of 0.8 ml for 8minutes. The reaction was quenched by the addition of EDTA to aconcentration of 5 mM and the solution was desalted using a Qiaquick PCRpurification column. Nicks and gaps were repaired by incubating at 12°C. for 30 minutes in a total volume of 90 μl in the presence of T4 DNApolymerase (6 units) and T4 DNA ligase (12 Weiss units) in the presenceof 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/mlBSA and 125 μM dNTPs. The DNA was desalted as before and the linear DNA(corresponding to the randomly circularly permuted bla) was isolatedfrom circular forms by agarose gel electrophoresis using the Qiaquickgel purification kit.

Random Domain Insertion

Plasmid pDIM-C8MalE has the malE gene encoding MBP under the IPTGinducible tac promoter. Introduction of a random double stranded breaks(one per molecule of pDIM-C8MalE) was performed as described (Spencer etal. 1993). One hundred ng of randomly linearized plasmid pDIMC8-MalE wasligated to 85 ng of randomly circularly permuted BLA fragment (5:1insert/vector molar ratio) in a reaction volume of 15 μl. The ligationwas carried out at 22° C. overnight in the presence of ligase buffer and45 Weiss units T4 DNA ligase. After ethanol precipitation, the ligatedDNA was transformed into Electromax DH5α-E electrocompetent cells byperforming ten electroporations of 40 μl cells each. Cells were platedon two 245×245 mm LB agar plates supplemented with 50 μg/mlchloramphenicol and incubated at 37° C. overnight. The naïve library wasrecovered from the large plates and stored in frozen aliquots asdescribed (Picard 2000).

Library Selection and Screening

The naïve library was plated on LB agar plates supplemented with 200μg/ml ampicillin and 50 mM maltose and incubated at 37° C. overnight.From these plates, 1056 colonies were picked to inoculate 1 ml LB media(supplemented with 50 μg/ml chloramphenicol and 1 mM IPTG) in 96-wellformat. After incubation overnight at 37° C., each culture was lysedusing 0.1 ml Popculture reagent, 40 units of rLysozyme, and 2.5 units ofbenzonase nuclease. Lysates were centrifuged to pellet the insolublematerial and the soluble fractions were assayed in 96-well format fornitrocefin hydrolysis in the presence or absence of 5 mM maltose using acolorimetric assay for nitrocefin hydrolysis (Posey et al. 2002). Theassays were carried out at room temperature using the Spectramax-384Plus microplate reader (Molecular Devices) in the presence of 100 mMsodium phosphate buffer and 50 μM nitrocefin in a 200 μl reactionvolume. Clones whose lysates exhibited a greater than 2-fold increase inthe rate of nitrocefin hydrolysis were recultured and their lysatesassayed again to verify the effect.

Protein Modifications and Mutagenesis

A GGSGH₉ sequence (SEQ ID NO: 77) was appended to the sequence of RG13by PCR amplification with the appropriate primers. The PCR product wascloned between NdeI and XhoI sites of pET24b (Novagen) to createpET24b-RG13. Mutations I329W and A96W were introduced into pET24b-RG13by a combination of overlap extension PCR and Quickchange mutagenesis.

Protein Purification

One liter LB media containing 50 μg/ml kanamycin was inoculated with 2%overnight culture and shaken at 37° C. The culture was induced with 1 mMIPTG when the OD₆₀₀ reached 0.5 and incubated at 22° C. for 16 hours.Pelleted cells were resuspended in 20 ml binding buffer supplied by theHis-tag protein purification kit (Novagen, Madison, Wis.) and lysed byFrench press. The soluble fraction was recovered and the protein waspurified using the protein purification kit. Eluted protein was dialyzedat 4° C. against three liters of 100 mM sodium chloride, 50 mM sodiumphosphate buffer overnight followed by dialysis against one liter of thesame buffer with 20% glycerol for four hours. Protein was stored inaliquots at −80° C. Fusion proteins RG13 and RG13(I329W) were purifiedas described above. To improve the yield of RG13(A96W/I329W), 10 mMmaltose was added to the culture at induction. RG13(A96W/I329W) wasdialyzed more extensively after purification and complete removal ofmaltose was verified by enzymatic assay on successive rounds of dialysisin the presence and absence of maltose. The purities of the proteinswere estimated by Coomassie blue staining of SDS-PAGE gels. The puritiesof RG13, RG13(I329W), and RG13(A96W/I329W) were greater than 98%, 95%and 97%, respectively. The extinction coefficients of RG13, RG13(I329W),and RG13(A96W/I329W) at 280 nm were calculated (Saghatelian et al. 2003)to be 126,000; 120,500 and 116,100 Abs M⁻¹ cm⁻¹, respectively.

Steady State Kinetics

All kinetic assays were performed at 25° C. in the presence of 100 mMsodium phosphate buffer, pH 7.0. Ten μl of enzyme stock was added to1.59 ml buffer (containing the saccharide, if desired). After incubationfor 30 seconds, 0.4 ml of 5× substrate was added and the absorbance atthe appropriate wavelength was recorded using the Cary50 UV-VISspectrophotometer. The wavelength monitored was 486 nm, 240 nm, and 232nm for nitrocefin, carbenicillin, and ampicillin respectively. From theinitial rate of reaction the kinetic constants were determined usingEadie-Hofstee plots. In the absence of maltose, the time course of thereaction for RG13, RG13(I329W), and RG13(A96W/I329W) displayed a slightlag in the reaction rate that became more pronounced at higher substrateconcentrations. The rate data was consistent with a small hystereticeffect (Brennan et al. 1994) and not substrate inhibition aspreincubation of the enzyme with the substrate for one minute preventedthe lag from occurring upon addition of more substrate. Therefore, thesteady state parameters for nitrocefin hydrolysis in the absence ofmaltose were determined by measuring the rate at 1-2 minutes (well afterthe lag) and correcting the substrate concentration by subtracting theamount of substrate hydrolyzed. In all cases the extent of reaction atthe point the rate was measured was less than 25%. In the presence ofmaltose, no lag was observed.

Maltose Affinity

Maltose affinity for RG13 (in presence and absence of 10 mMcarbenicillin) and RG13(I329W) (in the absence of substrate) wasdetermined using intrinsic protein fluorescence measured on a PhotonTechnology QuantaMaster QM-4 spectrofluorometer. Fluorescence spectrawere obtained at 25° C. at different concentrations of maltose in 50 mMsodium phosphate buffer, pH 7.0, containing 100 mM sodium chloride. Theprotein concentration was 50-100 nM. Excitation was at 280 nm. Thequenching in fluorescence intensity at 341 nm caused by maltose was usedin Eadie-Hofstee equivalent plots to determine K_(d) using the followingequation:

${\Delta\; F} = {{\Delta\; F_{\max}} - {K_{d}\frac{\Delta\; F}{\lbrack L\rbrack}}}$where ΔF is the change in fluorescence intensity, ΔF_(max) is thedifference in fluorescence between no maltose and saturating amounts ofmaltose and [L] is the maltose concentration. The fluorescence quenchingof RG13(I329W/A96W) upon addition of maltose was insufficient toaccurately determine a K_(d) by this method. The dissociation constantfor maltose and RG13(I329W) in the presence of saturating carbenicillin(2 mM) was determined by measuring the initial rate of carbenicillinhydrolysis as a function of maltose concentration. The apparentdissociation constant in the presence of subsaturating concentrations ofnitrocefin (25 μM) for all three proteins was determined by measuringthe initial rate of nitrocefin hydrolysis as a function of maltoseconcentration.

In Vivo Characterization of Switches

Overnight inoculums of DH5α-E cells expressing RG13, BLA or BLA(W208G)were diluted into LB media and plated on LB plates, either in theabsence or presence of 50 μM maltose, in the presence of increasingamounts of ampicillin. Ampicillin was present in the plates at thefollowing concentrations: 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, and2000 μg/ml. Cells were plated at approximately 1000 CFU (no antibiotic)per plate. The plates were incubated at 37° C. for 20 hours. The minimuminhibitory concentration (MIC) was defined as the lowest ampicillinconcentration at which no colonies were present, or that at which thenumber of colonies present was <1% of the number of colonies at the nextlowest level of ampicillin.

Characterization of BLA-MBP Molecular Switches

As discussed, the approach to construction of a model molecular switchinvolved recombination of the genes encoding TEM-1 β-lactamase (BLA) andthe E. coli maltose binding protein (MBP). BLA and MBP lack anysequence, structural or functional relationship except for the fact thatthey are periplasmic proteins of bacterial origin. BLA is a monomericenzyme that hydrolyzes the amide bond of the β-lactam ring of β-lactamantibiotics. The presence of maltose has no effect on wild type BLAenzymatic activity, with or without the presence of an equimolar amountof MBP (Guntas et al. 2004). MBP is a member of the periplasmic bindingprotein superfamily and is involved in chemotactic response and thetransport of maltodextrins. MBP consists of a single polypeptide chainthat folds into two domains connected by a hinge region. The singlebinding site for maltose is at the interface of these two domains. Inthe absence of maltose, MBP exists in an open form. Maltose-binding isconcomitant with a 35° bending motion about the hinge resulting in theclosed form of the protein (Sharff et al. 1992).

We sought to create a molecular switch by combining BLA and MBP in sucha manner that the rate of β-lactam hydrolysis was coupled to maltosebinding and maltose concentration. We reasoned that in such a switch theconformational change in the MBP domain upon maltose binding wouldpropagate to the active site of the BLA domain and alter its catalyticproperties, a mechanism analogous to natural allosteric effects.

The fragment of the BLA gene coding for the mature protein wascircularly permuted in a random fashion (Graf et al. 1996; Ostermeier etal. 2001) and subsequently randomly inserted into a plasmid containingthe E. coli malE gene that codes for MBP. FIG. 6A is a schematic diagramshowing the strategy used to make the molecular switch. Moreparticularly, FIG. 6A shows that the fragment of the BLA gene coding forthe mature protein (codons 24-286) is flanked by sequences coding for aGSGGG linker (SEQ ID NO: 1) (each of which contains a BamHI site). Thefragment is excised by digestion with BamHI and cyclized by ligationunder dilute DNA concentrations. A single, randomly-located doublestrand break is introduced by DNaseI digestion to create the circularlypermuted library. This library is randomly inserted into plasmidpDIMC8-MBP containing the MBP gene (malE) under control of the tacpromoter (tacP/O). The site for insertion in pDIMC8-MBP is created byintroduction of a randomly located double-stranded break by digestionwith dilute concentrations of DNaseI.

For the random circular permutation of bla [24-286], we fused the 5′ and3′ ends by an oligonucleotide sequence that would result in a GSGGGflexible peptide linker (SEQ ID NO: 1) between the original N- andC-termini of the protein. This linker was designed to be of sufficientlength to connect the termini without perturbing BLA structure.

For the random circular permutation of bla [24-286], we fused the 5′ and3′ ends by an oligonucleotide sequence that would result in a GSGGGflexible peptide linker between the original N- and C-termini of theprotein. This linker was designed to be of sufficient length to connectthe termini without perturbing BLA structure.

Statistical analysis on the resulting library indicated that a minimumof 27,000 members contained a circularly permuted bla[24-286] insertedinto malE in the correct orientation with both fusion points in-framewith malE. Approximately 0.33% of these members were able to formcolonies on rich media plates containing 200 μg/ml ampicillin and 50 mMmaltose. These library members were screened in 96-well format for amaltose dependence on β-lactamase activity using a colorimetric assayfor nitrocefin hydrolysis.

We identified one protein (RG13; FIG. 6B) in which the initial velocityof nitrocefin hydrolysis (at 50 μM nitrocefin) increased by 17-fold inthe presence of maltose. FIG. 6B is a schematic illustration of thesequence of the RG13 switch. The numbers in parentheses indicate theamino acid number of the starting proteins. The numbering system for MBPdoes not include the signal sequence. The numbering system for BLA doesinclude the signal sequence and does not follow the consensus numberingsystem for β-lactamases.

Referring to FIG. 6C, it was determined that in RG13, the BLA wascircularly permuted in a loop that precedes a β-sheet that lines theactive site of the enzyme. The circular permuted BLA was inserted at thebeginning of an α-helix of MBP such that two MBP residues were deleted.More particularly, FIG. 6C shows structures of maltose-bound MBP (Quichoet al. 1997) and BLA bound to an active-site inhibitor (Maveyraud et al.1996) oriented such that the fusion sites in RG13 are proximal.

Using purified RG13, we confirmed that the increase in catalyticactivity occurred only in the presence of sugars that are known to bindand induce a conformational change in MBP (FIG. 7). FIG. 7A shows thepercent increase in the initial velocity of nitrocefin hydrolysis at 20μM nitrocefin upon addition 5 mM of the indicated ligands (maltose,maltotriose and β-cyclodextrin) and non-ligands (sucrose, lactose andgalactose). It is seen that sugars known to induce a largeconformational change (Quicho et al. 1997) (i.e., maltose andmaltotriose; 35° closure angle) produced a 15- to 20-fold increase inthe rate of nitrocefin hydrolysis. β-cyclodextrin, which only induces a10° hinge bending motion in MBP (Evenas et al. 2001), increased the rate2-fold. Non-ligands such as sucrose, lactose and galactose had noeffect.

We next determined that the switching was reversible (i.e., uponremoving maltose, the activity returned to its pre-maltose level). Thiswas first demonstrated by competing bound maltose off RG13 usingβ-cyclodextrin (FIG. 7B). FIG. 7B shows reversible switching using thecompeting ligand. During the enzymatic hydrolysis of nitrocefin,formation of product was monitored by absorbance at 486 nm. At time zerothe reaction was started in 2 ml phosphate buffer (0.1M) with 20 μMnitrocefin and 2.5 nM RG13. At the time indicated by the first arrow, 20μl of 1M maltose was added resulting in a 10-fold increase in thereaction rate. This maltose concentration is above the K_(d) for maltosebut is subsaturating. At the time indicated by the second arrow, 230 μlof 10 mM β-cyclodextrin was added (final concentrations are 1.0 mMβ-cyclodextrin and 8.9 μM maltose). Because RG13 has similar affinitiesfor maltose and β-cyclodextrin but β-cyclodextrin is present ata >100-fold higher concentration, the β-cyclodextrin preferentiallyreplaces the maltose bound to RG13 and the rate of reaction decreases toa level consistent with β-cyclodextrin's modest effect on nitrocefinhydrolysis.

Reversibility of the switch was also demonstrated by subjecting RG13 torepeated rounds of dialysis and addition of maltose to cycle between lowand high levels of enzymatic activity. FIG. 7C shows reversibleswitching after dialysis. The initial rate of nitrocefin hydrolysis at25 μM nitrocefin was measured at the indicated steps. Maltose was addedto a final concentration of 5 mM.

This demonstrated reversibility is one of the features thatdifferentiates our approach from methods such as conditional proteinsplicing (Mootz et al. 2002; Buskirk et al. 2004) that producenon-reversible switches that control the production of active proteinrather than activity of the protein per se.

From steady state kinetics experiments, we determined theMichaelis-Menten parameters of RG13 for nitrocefin hydrolysis at 25° C.in the absence and presence of maltose. In the absence of maltose, thecatalytic constants were k_(cat)=200±40 s⁻¹ and K_(m)=550±120 μM. Withthe addition of saturating amounts of maltose, k_(cat) increased 3-foldand K_(m) decreased 8-fold, resulting in a 25-fold increase ink_(cat)/K_(m). The kinetic constants of RG13 in the presence ofsaturating concentrations of maltose (k_(cat)=620±60 s⁻¹ and K_(m)=68±4μM) were comparable to that previously reported for BLA at 24° C.(k_(cat)=900 s⁻¹ and K_(m)=110 μM (Sigal et al. 1984)). This findingshows that RG13 is a very active TEM1 β-lactamase in the presence ofmaltose. RG13 has exhibited switching behavior with all seven BLAsubstrate tested to date including ampicillin (16-fold rate increase at50 μM ampicillin) and carbenicillin (12-fold rate increase at 50 μMcarbenicillin).

The increase in k_(cat) indicates that maltose binding affects thecatalytic steps. However, since K_(m) is a combination of the rateconstants for substrate binding as well as catalysis (Christensen et al.1990), K_(m) could not be directly used to ascertain the effect ofmaltose on substrate binding. Instead, the effect of maltose onsubstrate binding was determined indirectly by measuring the effect ofsubstrate on maltose binding using intrinsic protein fluorescence. Thesestudies suggested that RG13 undergoes a conformational change much likeMBP does upon maltodextrin binding, since maltose-induced quenching oftotal fluorescence (˜10%) and shifting of the maximum fluorescencewavelength (i.e., a 1.5 nm red-shift for maltose and a 4 nm blue-shiftfor β-cyclodextrin) were similar to that previously reported for MBP(Hall et al. 1997). The presence of saturating amounts of the substratecarbenicillin decreased the dissociation constant of maltose and RG13from 5.5±0.5 μM to 1.3±0.5 μM. Thus, maltose binding must decrease thedissociation constant of carbenicillin and RG13 by the same factor (FIG.7).

FIG. 8 is a schematic diagram depicting coupling of ligand and substratebinding. More particularly, FIG. 8 shows that the change in free energyupon protein (P) binding ligand (L) and substrate (S) is the samewhether the ligand or substrate binds first. Adding the free energychanges of the two different paths from L+P+S to LPS, it is seen that:ΔG_(L)+ΔG_(S) ^(L)=ΔG_(S)+ΔG_(L) ^(S) since the total free energy changeis path independent. By rearranging this equation to: ΔG_(L)−ΔG_(L)^(S)=ΔG_(S)−ΔG_(S) ^(L) it is seen that the left hand side representsthe effect that the presence of bound substrate has on ligand bindingand the right hand side represents the effect that the presence of boundligand has on substrate binding. The effects must be equal. Thiscorresponds to a coupling energy of approximately 1 kcal/mol. Withoutintending to be bound by theory, this observation offers an additionalexplanation for the increase in β-lactam hydrolysis in the presence ofmaltose: a positive heterotropic allosteric effect on substrate binding.

Presumably, the BLA domain of the apo, open form of RG13 exists in acompromised, less active conformation. In the ligand-bound state, theBLA domain exists in a more normal, active conformation. We sought todetermine the state of the BLA domain in the process of closing. Weinvestigated at what closure angle the catalytic properties of RG13improved To address these questions, we took advantage of mutations inthe hinge region of MBP that manipulate the conformational equilibriabetween the open and closed state (Marvin et al. 2001). Residual dipolarcouplings have been used to establish that the apo forms of thesemutants are partially closed relative to the apo wildtype MBP with theensemble average closure angles being 9.5° and 28.4° for I329W andI329W/A96W, respectively (Millet et al. 2003). The ligand-bound closedforms of MBP, i.e., MBP(I329W) and MBP(I329W/A96W) have closure anglesof 35°. Partial closing shifts the equilibrium towards the ligand-boundstate and thus the mutations increase the affinity for maltose (Marvinet al. 2001).

Introduction of these mutations into RG13 resulted in the creation ofmore sensitive switches—i.e., switches that respond to lowerconcentrations of maltose (FIG. 9). FIG. 9A shows dissociation constantsfor maltose determined in the absence (white bars) and presence (blackbars) of saturating concentrations of carbenicillin. The apparentdissociation constants in the presence of subsaturating concentrations(25 μM) of nitrocefin (grey bars) were also determined. The dissociationconstants for maltose of MBP, MBP(I329W), MBP(I329W/A96W) (dashed line)reported by Marvin and Hellinga (2001) are shown for comparison (FIG.9A).

Without intending to be bound by theory, the fact that we observedqualitatively similar changes in maltose affinity when the mutations areintroduced into RG13 strongly suggests that the relative order andmagnitude of the angles of closure of RG13, RG13(I329W) andRG13(I329W/A96W) are similar to that of MBP, MBP(I329W) andMBP(I329W/A96W). Thus, the apo forms of the two RG13 mutants offerconformations intermediate between the open to the closed form ofRG13—conformations that may reflect that of RG13 in the process ofclosing. Assuming that the process of closing in RG13 passes through theconformations of the apo forms of the two RG13 mutants, kineticcharacterization of RG13(I329W) and RG13(I329W/A96W) suggested that theinitial stages of closing do not result in changes in the BLA domainthat substantially affect catalysis.

FIGS. 9B-D show steady state kinetic parameters of nitrocefin hydrolysisfor RG13, RG13(I329W) and RG13(I329W/A96W) in the presence (black bars)or absence (white bars) of saturating concentrations of maltose.Experimental conditions were as follows: 100 mM sodium phosphate buffer,pH 7.0, 25° C. Both k_(cat) and K_(m) improved during the intermediatestages of closing, but the majority of the effect on K_(m) occurredduring the final stages of closing.

As the magnitude of the allosteric effect was on the same order as thatof many natural allosteric enzymes, we next examined the biologicaleffects of RG13. We found that the switching activity was sufficient toresult in an observable phenotype: maltose-dependent resistance toampicillin (Table 8). E. coli cells expressing RG13 had a minimuminhibitory concentration (MIC) for ampicillin that was increasedfour-fold in the presence of 50 μM maltose. In contrast, the addition ofthe same concentration of sucrose or glucose to a plate did not affectthe MIC (Table 8). Thus, RG13 serves to couple the previously unrelatedfunctions of ampicillin

TABLE 8 Ampicillin Resistance of E. coli Cells in the Presence andAbsence of Maltose. Minimum Inhibitory Concentration of ExpressedAmpicillin (μg/ml)* Protein No maltose 50 μM maltose none 4 4 RG13 128512 BLA(W208G)† 32 32 BLA ≧2000 ≧2000 *Conditions: DH5α-E cells on LBplates (with or without maltose) incubated at 37° C. for 20 hours. †Amutant of BLA with reduced activity.resistance and maltose concentration. E. coli cells expressing RG13function as a growth/no growth sensor for maltose.

We have shown herein that two unrelated proteins can be systematicallyrecombined in order to link their respective functions and createmolecular switches. A combination of random circular permutation andrandom domain insertion enabled the creation of a MBP-BLA fusiongeometry in which conformational changes induced upon maltose bindingcould propagate to the active site of BLA and increase BLA enzymaticactivity up to 25-fold. The functional coupling of two proteins with noevolutionary or functional relationship is a powerful strategy forengineering novel molecular function. For example, coupling aligand-binding protein and a protein with good signal transductionproperties would result in the creation of a molecular sensor for theligand. Furthermore, switches that establish connections betweencellular components with no previous relationship can result in novelcellular circuitry and phenotypes. As discussed above, we expect suchswitches to establish connections between molecular signatures ofdisease (e.g., abnormal concentrations of proteins, metabolites,signaling or other molecules) and functions that serve to treat thedisease (e.g., delivery of drugs, modulation of signaling pathways ormodulation of gene expression) and therefore possess selectivetherapeutic properties.

Example 3 Design Considerations and Properties of Molecular Switches

This Example describes design considerations, kinetic properties andcharacteristics of families of molecular switches that can beconstructed according to the methods of the invention.

Molecular switch RG13, described above, has a dissociation constant formaltose of about 5-6 μM in the absence of a BLA substrate. In thepresence of saturating amounts of the substrate carbenicillin, thedissociation constant for maltose decreases to about 1 μM. This showsthat the binding of maltose and substrate (carbenicillin) are coupled.The coupling energy is on the order of 1 kcal/mol. This is consistentwith a decrease in K_(m) for nitrocefin in the presence of maltose (SeeTables 9 and 10, supra)

Switches Responding to a Range of Signal Concentrations

It is believed that a switch is most useful if the range of theconcentration of the signal (maltose, in the case of RG13) overlaps withthe range of signal concentration that the dependent function respondsto. When a ligand-binding protein is used as the signal detector and theligand is the signal, the latter range corresponds approximately to therange 0.1K_(d)-10 K_(d), where K_(d) is the dissociation constant of theswitch and the signal. This can be seen from the following example.

In the case of RG13, the velocity of nitrocefin hydrolysis is thedependent function. The velocity (v) of nitrocefin hydrolysis depends onthe steady state kinetic parameters by the Michaelis-Menten (Equation1).

$\begin{matrix}{v = \frac{{\lbrack E\rbrack_{0}\lbrack S\rbrack}k_{cat}}{K_{m} + \lbrack S\rbrack}} & (1)\end{matrix}$where [E]₀ is the concentration of the switch, [S] is the concentrationof nitrocefin and k_(cat) and K_(m) are the Michaelis-Menten kineticparameters. In the absence of maltose, the velocity is found by Equation2

$\begin{matrix}{v^{-} = \frac{{\lbrack E\rbrack_{0}\lbrack S\rbrack}k_{cat}^{-}}{K_{m}^{-} + \lbrack S\rbrack}} & (2)\end{matrix}$where the superscript “−” designates that the parameters are those whenmaltose is not bound to the switch. In the presence of saturatingconcentrations of maltose (i.e. maltose is bound to all switches; thisoccurs at very high concentrations of maltose relative to thedissociation constant K_(d) for maltose), the velocity is found byEquation 3:

$\begin{matrix}{v^{+} = \frac{{\lbrack E\rbrack_{0}\lbrack S\rbrack}k_{cat}^{+}}{K_{m}^{+} + \lbrack S\rbrack}} & (3)\end{matrix}$where the superscript “+” designates that the parameters are those whenmaltose is bound to the switch. At intermediate concentrations ofmaltose, the velocity depends on the fraction of switches that havemaltose bound. If we make the approximation that the small cooperativeeffect of maltose- and substrate-binding can be ignored, the fraction Fof switches that are bound to maltose can be found by Equation 4:

$\begin{matrix}{F = \frac{\lbrack M\rbrack}{\left\lbrack K_{d} \right\rbrack + \lbrack M\rbrack}} & (4)\end{matrix}$where [M] is the concentration of maltose. The velocity of nitrocefinhydrolysis is thus found by Equation 5:

$\begin{matrix}{v = {{F\frac{{\lbrack E\rbrack_{0}\lbrack S\rbrack}k_{cat}^{+}}{K_{m}^{+} + \lbrack S\rbrack}} + {\left( {1 - F} \right)\frac{{\lbrack E\rbrack_{0}\lbrack S\rbrack}k_{cat}^{-}}{K_{m}^{-} + \lbrack S\rbrack}}}} & (5)\end{matrix}$

Equation 5 is true for all concentrations of maltose as it reduces toEquations 2 and 3 in the limiting cases of no maltose bound andsaturating maltose, respectively. The fold-increase in the rate ofnitrocefin velocity Z is found by dividing the right hand side ofEquation 5 by the velocity in the absence of maltose to get Equation 6:

$\begin{matrix}{Z = {{F\frac{k_{cat}^{+}\left( {K_{m}^{-} + \lbrack S\rbrack} \right)}{k_{cat}^{-}\left( {K_{m}^{+} + \lbrack S\rbrack} \right)}} + \left( {1 - F} \right)}} & (6)\end{matrix}$

Referring to FIG. 10, Equation 6 is plotted for the case of RG13hydrolysis of 25 μM nitrocefin using a range of different dissociationconstants for maltose. More particularly, FIG. 10 shows the foldincrease in velocity of switch RG13 with different dissociationconcentrations (K_(d)) for maltose. The concentration of nitrocefin was25 μM. The kinetic parameters of RG13 with and without maltose are thoseshown in Table 9. Equation 6 was used to generate the curves. It isapparent that the velocity is changing most in the range of one order ofmagnitude higher or lower than the dissociation constant for maltose.The switch is expected to have the largest change in the dependentfunction if the concentration of the signal (maltose in the case ofRG13) changes within this range or changes through this range. Thus, itis desirable for the application of molecular switches to createswitches with different affinities for the signal so as to be useful fordifferent concentration ranges of the signal.

Altering Affinity for Signals

Exemplary switches were created by the method having differentaffinities for maltose. For example switch RG-5-169 (sequenceMBP[1-338]-BLA[34-286]-GSGGG-BLA[24-29]-MBP[337-370] (“GSGGG” disclosedas SEQ ID NO: 1) was created having a K_(d) for maltose (>1 mM) that ismuch greater than that of RG13 for maltose (1-5 μM).

The affinity of switches for effectors (signals) can also be altered bya variety of methods, including rational design and directed evolutionmethods. As long as the resulting altered-affinity switch maintains aconformational change upon binding the effector that results in changesthe dependent function, switching will be maintained. For example,mutations known to alter the affinity of the ligand recognition domain(for RG13 this is MBP) can be introduced into the switch to createswitches with altered affinity for the ligand. These mutations consistof those that make direct contact with the ligand, those that makecontact with residues that make direct contact with the ligand and thosethat are more distal from the binding site pocket.

For instance, as discussed in Example 2, mutations have been made in thehinge region of MBP that manipulate the conformational equilibriabetween the open and closed state (Marvin and Hellinga 2001). Residualdipolar couplings have been used to establish that the apo forms ofthese mutants are partially closed relative to the apo wildtype MBP withthe closure angles being 9.5° and 28.4° for I329W and A96W/I329W,respectively (the ligand-bound closed form of MBP has a closure angle of35°) (Millet, Hudson et al. 2003). Because partial closing shifts theequilibrium towards the ligand-bound state, the I329W mutation resultsin about a 20-fold increase in affinity for maltose and the A96W/I329Wdouble mutant results in a 60-fold increase in the affinity for maltosecompared to wildtype MBP at 25° C. (Marvin and Hellinga 2001). Theaffinities of MBP, MBP(I329W) and MBP(I329W/A96W) are 800 nM, 35 nM and13 nM, respectively.

Introduction of the above MBP mutations into RG13 resulted in mutantswith increased affinity for maltose (Table 9) while still maintainingswitching behavior (Table 10). In addition, the level of activity in thepresence of saturating amounts of maltose (the “on” state) was notaffected by the mutations (Table 10).

TABLE 9 Maltose Affinity of RG13-Based Molecular Switches. K_(d) maltose(μM)^(a) Saturating Carbenicillin No 25 μM Enzymatic Protein LigandSubstrate^(b) nitrocefin^(c) IPF^(b) assay^(d) RG13 maltose 5.5 ± 0.56.7 ± 0.03 1.3 ± 0.5 0.9 ± 0.1 RG13 maltose 0.55 ± 0.13 1.0 ± 0.04 nd0.11 ± 0.01 I329W RG13 maltose nd 0.17 ± 0.02  nd nd I329W/ A96W^(a)Conditions: 100 mM NaCl, 50 mM NaPO₄, pH 7.0, 25° C. ^(b)Determinedby measuring intrinsic protein fluorescence (IPF) as a function ofmaltose concentration. When using IPF at saturating carbenicillin, aconcentration of 10 mM carbenicillin was used. ^(c)Determined bymeasuring the initial rate of nitrocefin hydrolysis as a function ofmaltose concentration. 25 μM nitrocefin is well below the K_(m) ofnitrocefin. Thus, most molecules of RG13 will not have nitrocefin boundand the effective K_(d) that is measured is close to what it would be inthe absence of substrate. ^(d)Determined by measuring the initial rateof carbenicillin hydrolysis as a function of maltose concentration. Aconcentration of 1.5 mM carbenicillin was used, which is well above theK_(m) of carbenicillin. Thus, most molecules of RG13 will havecarbenicillin bound and the K_(d) that is measured is in the presence ofbound substrate (carbenicillin).

TABLE 10 Kinetic Parameters of Nitrocefin Hydrolysis^(a) of RG13-BasedMolecular Switches. k_(cat) K_(m) k_(cat)/K_(m) ^(a) k_(cat)/K_(m)Protein Effector k_(cat) (s⁻¹)^(a) Ratio^(b) K_(m) (μM)^(a) Ratio^(b)(s⁻¹ μM⁻¹) Ratio^(b) RG13 — 200 ± 40 —  550 ± 120 0.37 ± 0.10 RG13 I329W— 190 ± 30 — 350 ± 60 0.54 ± 0.11 RG13 — 360 ± 40 — 260 ± 40 1.4 ± 0.3I329W/A96W RG13 maltose 620 ± 30 3.1 ± 0.6 68 ± 4 0.12 ± 0.03 9.2 ± 0.725 ± 7  RG13 I329W maltose 590 ± 50 3.1 ± 0.5 53 ± 7 0.15 ± 0.03 11.0 ±1.8  20 ± 5  RG13 maltose 530 ± 20 1.5 ± 0.2 60 ± 4 0.23 ± 0.04 8.9 ±0.8 6.4 ± 1.3 I329W/A96W RG13 β-cyclo^(c) 590 ± 60 2.9 ± 0.6 870 ± 901.6 ± 0.4 0.67 ± 0.10 1.8 ± 0.6 ^(a)Conditions: 100 mM sodium phosphatebuffer, pH 7.0, 25° C.; concentration of effector is 5 mM ^(b)(witheffector)/(without effector). ^(c)β-cyclodextrin

From a practical standpoint, the increase in maltose affinity of thesehinge mutants indicates that ligand-affinity of RG13 can besystematically changed to create molecular switches that respond todifferent concentration ranges of effector while still maintainingswitching ability and high activity in the presence of the effector. Byincreasing the affinity for maltose one increases the sensitivity of theswitch (i.e., it will switch to a higher level of activity at lowerconcentrations of maltose). Combinations of these affinity-alteredswitches are expected to behave as a composite switch with a highdynamic range.

Example 4 Modified Molecular Switches with Altered Signal Recognition

The invention further encompasses methods to alter the specificity ofthe signal recognition domain so that it recognizes other signals. Thisallows for the construction of “modified” molecular switches in whichthe dependent function responds to new signals without the need toconstruct entirely new molecular switches. For the example of RG13, inwhich the signal binding domain is the maltose binding protein, thesemethods can change the ligand to which the switch binds. This wouldallow the construction of molecular switches in which BLA activity couldbe modulated by different ligands. In one aspect of the method, theidentity of the signal to which the switch responds is altered byintroducing mutations into existing switches. For example, mutations inthe signal recognition domain already known to alter the ligand-bindingspecificity can be introduced into the switch to create switches thatrespond to new ligands. For instance, Hellinga and colleagues havecomputationally designed periplasmic binding proteins with radicallyaltered binding specificities (Looger, Dwyer et al. 2003) includingdesigning MBP to bind Zn²⁺ (Marvin and Hellinga 2001) instead ofmaltose. MBP binds maltose with high affinity (K_(d)=0.8 μM) but doesnot bind Zn²⁺. MBP with the A* set of mutations (A63H/R66H/Y155E/W340E)has high affinity for Zn²⁺ (K_(d)=5.1 μM) and does not bind maltose(Marvin and Hellinga 2001). Accordingly, introduction of the A* set ofmutations into a fusion such as RG13 may result in a switch thatresponds to Zn²⁺ but not maltose.

The signal recognition domain can be altered by rational design ordirected evolution to bind to new effectors. With respect to testingmutations predicted by rational design or screening or selectinglibraries created for a directed evolution approach, existing switchesare used to efficiently test or select for binding to new ligands invivo. For example, E. coli cells expressing the MBP-BLA switch RG13 fromthe lac promoter on pDIMC8 have a higher MIC for ampicillin (Amp) in thepresence of maltose than in their absence (Table 11) because the BLAenzymatic activity of RG13 (hydrolysis of ampicillin) is higher in thepresence of maltose. Thus, for example, mutations created in RG13(either by rational design or by a stochastic or semi-stochastic method)such that mutant forms of RG13 bind another ligand X (and behave as aswitch) can be screened or selected for in vivo. E. coli producing sucha new switch will grow at 200 μg/ml Amp in the presence of X but not inthe absence of X.

TABLE 11 Minimum Inhibitory Concentration of Ampicillin for E. coliCells Expressing RG13^(a). MIC ampicillin Supplement to plate (μg/ml)none 100 50 μM maltose 400  5 mM maltose 400 ^(a)conditions: LB plates,37° C., supplemented with maltose as indicated. Approximately 1000colony forming units (without ampicillin) per plate. Concentrations ofampicillin tested: 0, 25, 50, 100, 200, 400 and 800 μg/ml.

Ligands that bind to the signal recognition domain in a different mannerhave different switching ability. This is demonstrated by the fact thatβ-cyclodextrin, which is known to bind to MBP but with a differentconformational change (Skrynnikov, Goto et al. 2000; Hwang, Skrynnikovet al. 2001), changes the activity of the RG13 switch in a differentmanner than maltose (see Table 10).

Example 5 Creation of Libraries Containing Families of MolecularSwitches

This example describes several strategies, including use of iterativeapproaches, for producing various types of libraries that containfamilies of related molecular switches.

Materials and Methods MBP-BLA Library Constructions

Random domain insertion and random circular permutation of the bla genewere performed generally as described in Examples above. Librariesdesignated 2-5 and 7 (having inserts at a particular site in the MBPgene) were constructed as shown schematically in FIG. 11. (See alsoFIGS. 2 and 3 supra for details on construction of the circular blagene, and FIG. 4 for details on preparation of the MBP-containingplasmid.) FIG. 12 is a schematic diagram showing the construction ofLibrary 6, in which a specific circular permuted version of bla wasrandomly inserted into the plasmid containing the MBP gene. (See alsoFIG. 1, left side).

MBP-BLA Library Selection and Screening

Libraries were plated on LB plates containing 5 mM maltose at theindicated concentrations of ampicillin and incubated at 37° C.overnight. From these plates, colonies were picked to inoculate 1 ml LBmedia (supplemented with 50 μg/ml chloramphenicol and 1 mM IPTG) in96-well format. Lysates from these cultures were assayed for nitrocefinhydrolysis activity in the presence and absence of maltose as describedabove.

Protein Characterization

His-tagged proteins were purified as described in Examples above. Allenzymatic assays were performed in the presence of 100 mM sodiumphosphate buffer, pH 7.0. Enzyme stock was added to 1.9 ml buffer(containing the saccharide, if desired). After incubation at the desiredtemperature for 5 minutes, 0.1 ml of 20× substrate was added and theabsorbance at the appropriate wavelength was recorded using the Cary50UV-VIS spectrophotometer. The wavelengths monitored were as follows:nitrocefin (486 nm), carbenicillin (240 nm), ampicillin (235 nm),cefazolin (260 nm), cefotaxime (260 nm), and cephalothin (260 nm).Ligand affinity was determined as described above. The oligomeric stateof MBP317-347 was determined by analysis of size exclusionchromatography data using a pre-packed column of superose 6 (Pharmacia,Pscataway, N.J.) with a separation range of 5-5000 kDa on a PharmaciaFPLC system. The mobile phase was phosphate buffer at pH 7.0 (0.1Msodium phosphate, 0.15 M NaCl) with or without 5 mM maltose and flowrate was set at 0.5 ml/min Elution peaks were detected by UV absorbanceat 254 nm. The column was calibrated using ribonuclease A (13.7 kD),albumin (67 kD), aldolase (158 kD), catalase (232 kD) as molecularweight standards.

Characteristics of Libraries

Libraries 2-7 were plated on different levels of ampicillin in thepresence of 50 mM maltose. Colonies that grew were used to inoculate96-well plates. The resulting cultures were lysed and assayed at roomtemperature for nitrocefin hydrolysis in the presence and absence ofmaltose in 96-well format. Library members in which the addition ofmaltose resulted in a 2-fold or greater difference in the rate ofnitrocefin hydrolysis were chosen for further study. Statistics on alllibraries and screening can are shown in Tables 12-14.

TABLE 12 Library Statistics for Libraries 2-7. Table discloses “GSGGG”as SEQ ID NO: 1. Library size (number of Library transformants with blainsert). Library 2 (T164-165/DKS) 0.44 × 10⁶ Library 3 (T164-165/GSGGG)1.05 × 10⁶ Library 4 (EE/DKS) 1.03 × 10⁶ Library 5 (EE/GSGGG) 0.30 × 10⁶Library 6 0.75 × 10⁶ Library 7 1.16 × 10⁶

Table 13 shows the number of library members that could grow on platescontaining different amounts of maltose and ampicillin. Based in part onthis information, colonies from different plates were screened.

TABLE 13 Number of Original Transformants Capable of Growth In Presenceof Ampicillin Number of original transformants that could grow on . . .No Maltose 50 mM maltose Library Amp5 Amp50 Amp200 Amp1000 Amp5 Amp50Amp200 Amp1000 2 734 394 220 — 1098 515 182 — 3 878 294 — 74 761 361 24088 4 7052 1747 1080  — 8354 2414 1525 — 5 3510 1159 298 — 4056 1615 630— 6 — 3138 383 64 — 4439 765 65 7 — 2008 990 138  — 1806 1337 275 

The number of colonies screened from plates containing different amountsof maltose and ampicillin is shown in Table 14. Colonies were screenedas described in the Methods section. For Libraries 2-5, all switchesoriginated from plates with 50 mM maltose and 5 μg/ml ampicillin. ForLibraries 6 and 7, all switches originated from plates with 50 mMmaltose and 200 μg/ml ampicillin.

TABLE 14 Number of Colonies Screened. Number of colonies screened fromplates containing . . . No Maltose 50 mM maltose Library Amp5 Amp50Amp200 Amp1000 Amp5 Amp50 Amp200 Amp1000 2 — — — —  96 672  80 — 3  96 —— — 192 576 384 — 4 — — — — — 576 — — 5 288 — — — 864 768 — — 6 — — — —— — 576 192 7 — — — — — — 1056  —

Molecular Switches Isolated from BLA-MBP Libraries 2-7

FIG. 13 is a schematic depiction of the library construction schemes forLibraries 2-7, and of particular switches identified from theselibraries. The arrowheads indicate the sites of insertion. Multiplearrowheads on one gene indicate random insertion sites. Dashed arrowsindicate a particular switch on which successive libraries were based.The magnitude of switching was determined on the soluble fraction ofcell lysates at room temperature using 50 μM nitrocefin. For switcheswith a rate increase in the presence of maltose, the ratio is of “withmaltose” to “without maltose” (indicated by no sign in front of thevalue). For switches with a rate decrease with maltose, the rate is of“without maltose” to “with maltose” (indicated by a negative sign infront of the value).

Referring to FIG. 13, five new switches were identified with improvedswitching activity, including one (designated IFG277) in which maltosewas a negative effector. Another switch (designated IFD15) was permutedsuch that residues 168-170 of BLA were tandemly duplicated. Residues168-170 are part of the Ω-loop associated with the active site of theenzyme that includes a key catalytic residue, Glu166. IFD15 was not abetter switch than the other four identified from these libraries.However, the fact that BLA could be permuted so near the active sitewithout elimination of activity, combined with the notion that aconnection between BLA and MBP near the active site of BLA would be morelikely to produce switches with superior properties, led us to choosethis particular circular permutation of the bla gene for Library 6.

Library 6 contained this particular circularly permuted variation of blarandomly inserted into the gene for MBP (FIGS. 12, 13). From thislibrary several new switches were identified, including BLA168-89 inwhich 22 residues near the C-terminus of MBP were deleted. However, thebest switches found had BLA inserted in the region between residues 316to 320. BLA168-81, whose catalytic activity increased almost two ordersof magnitude in the presence of maltose, had the circular permuted BLAinserted in place of residue 317 of MBP. Interestingly, RG13 alsoconsists of an insertion in place of residue 317, but with a differentcircular permutation of BLA.

To exhaustively explore insertions of circular permuted variants of BLAthat replace residue 317 of MBP, Library 7 was constructed. Forselecting library members from Library 7 for further examination, acriterion of 30-fold or better difference in catalytic activity withmaltose was selected. Three switches with sequences very similar toBLA168-81 were identified from Library 7 (FIG. 13).

Characterization of Switches

A 10×-His tag was added to the C-terminus of switches MBP317-347,MBP317-639 and BLA168-81 and the proteins were purified to >95% purityvia nickel-affinity chromatography. The enzymatic activity of theswitches was characterized using the colorimetric substrate nitrocefin(FIG. 14). FIG. 14A shows hydrolysis of 80 μM nitrocefin by 27 nMMBP317-347 in the presence and absence of maltose at 25° C. Moreparticularly, the reaction was started by the addition of nitrocefin attime zero to samples lacking (solid lines) or containing (dashed line) 5mM maltose. For the reaction traced by the solid grey line, 5 mM maltosewas added to the reaction at about 6 minutes. As can be seen in FIG.14A, the rate of nitrocefin hydrolysis was profoundly affected bymaltose. Only sugars known to bind MBP were effectors; sucrose,galactose and lactose had no effect on the rate of hydrolysis.

In none of the three switches did enzymatic activity obeyedMichaelis-Menten kinetics. In the absence of maltose, catalysis wascharacterized by a small burst lasting on the order of several minutesfollowed by a slower steady state rate (FIG. 14B). FIG. 14B shows thesame data as FIG. 14A with a narrower range of absorbance shown. Thegrey line is the background rate of nitrocefin hydrolysis in the absenceof enzyme. The size of the burst was much greater than 1 mol product/molof enzyme and was consistent with a branched pathway mechanism involvingsubstrate induced progressive inactivation (Waley, S. G., 1991). Suchkinetics have been observed previously in class A β-lactamases onsubstrates with bulky side chain substituents (Citri et al., 1976) thatorient towards the Ω-loop (Chen et al., 1993; Strynadka et al., 1992) aswell as in mutants of Staphylococcus aureus PC1 β-lactamase in which theΩ-loop has been perturbed (Chen et al., 1999). Similar burst kineticswere seen in the presence of maltose; thus substrate-inducedinactivation cannot be an explanation for the compromised activity inthe absence of maltose.

Preliminary characterization indicated that switch MBP317-347 had thelargest switching activity, and this switch was characterized in moredetail. In order to get an effective measure of the difference incatalytic activity between with and without maltose, the amount of timenecessary to convert half of the substrate to product was characterizedas a function of switch concentration and nitrocefin concentration (FIG.14C). Because the catalytic activities with and without maltose differedso greatly, there was only a limited protein concentration range inwhich both activities could be measured. In this range, the amount oftime necessary to convert half the substrate to product was 240-590times greater in the absence of maltose than in its presence. Moreparticularly, FIG. 13C shows the time necessary for MBP317-347 toconvert half of the nitrocefin to product at 25° C. as a function ofnitrocefin concentration, maltose and MBP317-347 concentration. Squaresindicate 5 μM nitrocefin; circles indicate 100 μM nitrocefin; filledsymbols indicate with maltose; open symbols indicate without maltose.

Referring to FIG. 14D, it was seen that the effect of temperature andsubstrate on switching activity was complex, with no clear trend. FIG.14D shows the ratio of time necessary for MBP317-347 to convert half ofsubstrate to product in the absence of maltose to that in the presenceof maltose as a function of substrate and temperature. White barsindicate 25° C.; black bars indicate 37° C. Concentrations ofMBP317-347/concentration of substrate are: ampicillin (113 nM/200 μM),carbenicillin, (453 nM/200 μM), cefazolin (113 nM/200 μM), cefotaxime(453 nM/100 μM), cephalothin (226 nM/150 μM), and nitrocefin (22.6nM/100 μM) Interestingly, the effect of switching the temperature from25 to 37° C. was a uniform ˜2-fold decrease in activity in the presenceof maltose, whereas the effect in the absence of maltose ranged from a3.5-fold increase to a 23-fold decrease in activity.

The oligomeric state of switch MBP317-347 at 25° C. was investigatedusing size exclusion chromatography. This analysis was consistent with amonomer-dimer equilibrium with a dissociation constant of about 5 μM inthe absence of maltose and about 20 μM in the presence of maltose. Theimportance of the dimerization and its minor maltose-dependence to theswitching activity is likely minimal—the difference in activity betweenwith and without maltose does not have a significant dependence onprotein concentration (FIG. 14C) and all the protein concentrationsassayed are well-below the dissociation constant of the dimer.

Example 6 Creation of Molecular Switches Binding Novel Ligands

Creation of Ligand-binding Site Library in MBP317-347 (Library SB3)

A library of variants of MBP317-347 was constructed in which each of thecodons coding for the five positions (D14, K₁₅, W62, E111, and W230) wascompletely random. Five sets of primers (in which the above codons werevaried as 5′-NNK-3′) were used to amplify fragments of the MBP317-347gene. Sequences of primers for creating Library SB3 are as shown.

Primer set #1 DIMC8Malfor (SEQ ID NO: 76)5′-GGACCAGGATCCATGAAAATAAAAACAGGT-3′ MBP1415rev (SEQ ID NO: 26)5′-GCCGTTAATCCAGATTAC-3′ Primer set #2 MBP1415for (SEQ ID NO: 27)5′-GTAATCTGGATTAAGGCNNKNNKGGCTATAACGGTCTCGCT-3′ MBP62rev (SEQ ID NO: 28)5′-GAAGATAATGTCAGGGCC-3′ Primer set #3 MBP62for (SEQ ID NO: 29)5′-GGCCCTGACATTATCTTCNNKGCACACGACCGCTTTGGT-3′ MBP111rev (SEQ ID NO: 30)5′-AACAGCGATCGGGTAAGC-3′ Primer set #4 MBP111for (SEQ ID NO: 31)5′-GCTTACCCGATCGCTGTTNNKGCGTTATCGCTGATTTAT-3′ MBP230rev (SEQ ID NO: 32)5′-CGGGCCGTTGATGGTCAT-3′ Primer set #5 MBP230for (SEQ ID NO: 33)5′-ATGACCATCAACGGCCCGNNKGCATGGTCCAACATCGAC-3′ DIMC8Malback(SEQ ID NO: 34) 5′-ATCCGGACTAGTAGGCCTTTACTTGGTGATACGAGT-3′

These fragments were assembled into a full gene by overlap extension PCRin a single PCR reaction. The assembled gene library was insertedbetween the BamHI and SpeI sites of pDIM-C8 to create a library of1.58×10⁷ transformants

Selection and Screening of Library SB3

The library was plated on LB plates containing 256 μg/ml ampicillin andvarious amounts of sucrose as shown in Table 15. The number oftransformants in the original library that could grow under theseconditions was determined by the product of frequency of colonies thatgrew and the number of transformants in the library (1.575×10⁷).Individual colonies were screened as described in the Methods sectionfor the MBP-BLA libraries except that sucrose was used instead ofmaltose. The number of colonies screened from the different plate typesis shown in Table 15.

TABLE 15 Analysis of Library BS3. Sucrose on plate Quantity none 0.5 mM5 mM 50 mM Transformants that can grow 220 255 372 >886 on 256 μg/ml AmpColonies screened — 369 46 170

Switch MBP317-347, described above, conferred upon E. coli cells amaltose-dependent ampicillin resistance phenotype. The MIC at 37° C. forcells plated on media containing 5 mM maltose was 512 μg/ml, which wasfour-fold higher than the MIC on plates lacking maltose. Other sugars,including sucrose, had no effect on the MIC. The only four-folddifference in MIC was somewhat surprising considering the much largeeffect of maltose on β-lactam hydrolysis in vitro.

Switch MBP317-347 connects the presence of a ligand (i.e., maltose) to agrowth/no growth phenotype when cells producing MBP317-347 are plated onβ-lactam antibiotics. We sought to exploit this phenotype to createswitches that respond to new effectors (FIG. 15). We reasoned that ifthe maltose-binding site of the switch was altered such that it bound anew ligand, and if binding of this new ligand induced a similarconformational change in the switch, then the β-lactamase activity ofthe switch would increase to a higher level of activity. Thus, from alibrary in which the maltose-binding site of the switch was randomized,one could select for those members that bound a new ligand by plating inthe presence of the new ligand on plates containing a level of β-lactamantibiotic that was not permissive for growth in the absence of the oldligand. We also predicted that once mutations necessary to convert themaltose switch into one for the new ligand were identified, introductionof these mutations into MBP would result in a binding protein for thenew ligand (FIG. 15).

This was tested by attempting to convert MBP317-347 into a switch thatresponds to sucrose. Maltose is a disaccharide of glucose whereassucrose is a disaccharide of glucose and fructose. Neither MBP norMBP317-347 show any detectable binding of sucrose (K_(d)>>50 mM). Byinspection of the crystal structure of MBP bound to maltose, weidentified five residues proximal to the glucose that is replaced withfructose in sucrose: D14, K15, W62, E111, and W230. A library ofvariants of MBP317-347, in which each of the five positions wasrandomized using 5′-NNK-3′ for each codon, was created by overlapextension that consisted of 1.58×10⁷ transformants (with a theoreticaldegeneracy on the protein level of 4.08×10⁶). This library was plated at37° C. in the presence of 256 μg/ml ampicillin and increasingconcentrations of sucrose.

In the absence of sucrose, the frequency of library members that grewwas ˜1.6×10⁻⁵. We speculate that these false positives result frommutations that increase the production of the switch or alleviate thedeficiency in ampicillin hydrolysis in the absence of bound ligand. Thefrequency of colonies on plates with 500 μM sucrose was notstatistically different than that on plates with no maltose. However thefrequencies of colonies growing at 5 mM and 50 mM sucrose were ˜2.6×10⁻⁵and >6×10⁻⁵, respectively.

Colonies (arising from the first library) from plates containing 256μg/ml ampicillin containing 500 μM sucrose or 50 mM sucrose were used toinoculate 96-well plates. Lysates of these cultures were screened (usingthe 96-well nitrocefin assay) for those members for which the rate ofnitrocefin hydrolysis increased in the presence of 5 mM sucrose. Twolibrary members (designated 5-7 and 6-47) were found to respond tosucrose from the 500 μM sucrose plate (Table 15). Many library membersthat grew on the 50 mM sucrose plate were found to respond to sucrose.These were further screened for those that responded to lower levels ofsucrose resulting in the identification of two more sucrose switches(designated 1-59 and 1-68).

TABLE 16 Sequences, Ligand Affinity and Switching Activity of EngineeredProteins. K_(d) for ligand (μM) at 25° C. in presence of Amino acidnumber No substrate^(a) 5 μM nitrocefin^(b) Protein 14 15 62 111 230Sucrose Maltose Sucrose Maltose Switching^(c) MBP317- D K W E W nb^(d)0.5 ± 0.1 nb^(d)  1.9 ± 0.2 240  347 5-7 L F Y Y W 0.7 ± 0.1 23 ± 13 6.7 ± 0.2 35 ± 5 82 6-47 L Q Y Q W — — 220 ± 10  3.2 ± 0.3 91 1-59^(e)K E Y R W — — 340 ± 20 44 ± 2 28 1-68^(e) L E Y R W — — — — 32 SBP(5-7)L F Y Y W 6.6 ± 0.6 24 ± 4  n/a n/a n/a MBP D K W E W nb^(d) 1 n/a n/an/a Abbreviations; nb, no binding; n/a, not applicable ^(a)Dissociationconstants determined by change in intrinsic protein fluorescence as afunction of ligand concentration (Hall et al., 1997). ^(b)Apparentdissociation constants in the presence of nitrocefin were calculatedusing change in initial rates of nitrocefin hydrolysis as a function ofligand concentration². ^(c)Ratio (without ligand to with ligand) of timenecessary to hydrolyze one-half of the substrate (100 μM nitrocefin; 25°C.; 20 nM protein; saturating ligand concentration). The ligand used wassucrose except maltose was used for MBP317-347. For 1-59 and 1-68,ligand affinity and switching was determined in the soluble fraction ofcell lysates, so the exact protein concentration is unknown. ^(d)Nobinding can be detected. K_(d) >> 50 mM. ^(e)characterized in thesoluble fraction of cell lysates

Characterization of Sucrose Switches

A 10×-His tag was added to the C-terminus of switches 5-7 and 6-47,described above, and the proteins were purified to >95% purity vianickel-affinity chromatography. The binding to sucrose and to maltosewas characterized by two different methods. Intrinsic proteinfluorescence (Hall et al., 1997) was used to directly determine a K_(d)for the ligand. Switch 6-47 showed too little change in fluorescenceupon incubation with sucrose or maltose, presumably in part due to theW62Y mutation. An apparent K_(d) was estimated using the effect of theligand on the initial rate of nitrocefin hydrolysis (Guntas et al.,2004). This was performed at both low and high substrate to illustratehow the presence of bound nitrocefin has a negative effect on ligandbinding; thus, the presence of bound ligand has a negative effect onsubstrate binding. Since sucrose-binding results in an increase incatalytic activity, large increases in the rates of the catalytic stepsin the presence of sucrose must compensate for the decreased substrateaffinity.

All of the switches still retained significant maltose affinity, with5-7 being the switch with both the highest affinity for sucrose(K_(d)=0.7 μM) and the highest specificity for sucrose over maltose(33-fold higher affinity for sucrose). No binding or switching inresponse to lactose or galactose was observed. Sucrose and maltoseincreased β-lactamase activity of by equal amounts. However, theswitching magnitude (ratio of activity with and without maltose) wasless than that observed in the parental maltose switch MBP317-347. Thereasons for this were examined in 5-7. In the absence of either sucroseor maltose, 5-7's activity was about 3-fold higher than MBP317-347's.The measured activity of 5-7 and MBP317-347 in the presence of boundligand did not differ significantly. This suggests that the apo form of5-7 is less compromised than MBP317-347 in nitrocefin hydrolysisactivity and that the conformation of 5-7 bound to maltose or sucrose isthe same—at least as far as its effect on 5-7's β-lactamase activity.

Creation of a Sucrose Binding Protein (SBP)

The D14L, K₁₅F, W62Y and E111Y mutations of sucrose switch 5-7 wereintroduced into a His-tag version of MBP to create SBP. SBP was purifiedto >95% purity via nickel-affinity chromatography. The affinity of SBPfor maltose was the same as that of sucrose switch 5-7 but the affinityfor sucrose was decreased by about 10-fold. Still, SBP maintained a4-fold preference for sucrose. The conversion of MBP to SBP represents a>>10⁶ conversion in binding specificity.

Example 7 Exemplary Molecular Switches

This example provides nucleic acid and amino acid sequences of severalexemplary molecular switches according to the invention.

Switch BLA168-81: Nucleic Acid Sequence: (SEQ ID NO: 35)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 36)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKSwitch MBP 317-347: Nucleic Acid Sequence: (SEQ ID NO: 37)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGC CTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino Acid Sequence:(SEQ ID NO: 38) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK Switch MBP 317-639:Nucleic Acid Sequence: (SEQ ID NO: 39)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtCCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino Acid Sequence:(SEQ ID NO: 40) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTS KVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK Switch MBP 317-694:Nucleic Acid Sequence: (SEQ ID NO: 41)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcttttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino Acid Sequence:(SEQ ID NO: 42) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK Switch BLA168-88:Nucleic Acid Sequence: (SEQ ID NO: 43)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 44)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKEEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGR QTVDEALKDAQTRITKSwitch BLA168-45: Nucleic Acid Sequence: (SEQ ID NO: 45)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCcacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 46)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLINEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSwitch BLA168-69: Nucleic Acid Sequence: (SEQ ID NO: 47)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 48)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSwitch BLA168-86: Nucleic Acid Sequence: (SEQ ID NO: 49)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 50)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAAS GRQTVDEALKDAQTRITKSwitch BLA168-89: Nucleic Acid Sequence: (SEQ ID NO: 51)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 52)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKSucrose Switch 5-7: Nucleic Acid Sequence: (SEQ ID NO: 53)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcttgtttggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctatgcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgtttatgcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino Acid Sequence:(SEQ ID NO: 54) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGLFGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFYAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVYALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSucrose Switch 6-47: Amino Acid Sequence: (SEQ ID NO: 55)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGLQGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFYAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVQALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSucrose Switch 1-59: Nucleic Acid Sequence: (SEQ ID NO: 56)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcaaggagggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctatgcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttcgggcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 57)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGKEGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFYAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVRALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSucrose Switch 1-68: Nucleic Acid Sequence: (SEQ ID NO: 58)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcttggagggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctatgcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttcgtgcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGACAAGAGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAACTGAATGAAGCCgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 59)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGLEGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFYAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVRALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSwitch RG 13: Nucleic Acid Sequence: (SEQ ID NO: 60)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGGATCCGGCGGTGGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 61)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGSATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTV DEALKDAQTRITKSwitch RG13 I329W: Nucleic Acid Sequence: (SEQ ID NO: 62)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgaggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGGATCCGGCGGTGGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTccgccaccatggaaaacgcccagaaaggtgaaTGGatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 63)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGSATMENAQKGEWMPNIPQMSAFWYAVRTAVINAASGRQTV DEALKDAQTRITKSwitch RG13 I329W/A96W: Nucleic Acid Sequence: (SEQ ID NO: 64)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatTGGgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGGATCCGGCGGTGGCCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTccgccaccatggaaaacgcccagaaaggtgaaTGGatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 65)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDWVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGSATMENAQKGEWMPNIPQMSAFWYAVRTAVINAASGRQTV DEALKDAQTRITKSwitch IFD7: Nucleic Acid Sequence: (SEQ ID NO: 66)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggaactgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgggacaagagccacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino acid Sequence:(SEQ ID NO: 67) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTDGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKSwitch IFG277: Nucleic Acid Sequence: (SEQ ID NO: 68)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggcttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggggatccggcggtggccacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcagacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaa Amino Acid Sequence:(SEQ ID NO: 69) MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMN ADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKSwitch IFD15: Nucleic Acid Sequence: (SEQ ID NO: 70)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgggacaagagccacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggaactgaatgaagccgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagtaaAmino Acid Sequence: (SEQ ID NO: 71)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWDKSHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASG RQTVDEALKDAQTRITKSwitch EEG251: Nucleic Acid Sequence: (SEQ ID NO: 72)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggggatccggcggtggccacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggagagtactcaccagtcacagaaaagcatcttacggatggcaagtgaAmino Acid Sequence: (SEQ ID NO: 73)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQND LVEYSPVTEKHLTDGKSwitch EEG530: Nucleic Acid Sequence: (SEQ ID NO: 74)atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgccaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactcgtatcaccaagggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggggatccggcggtggccacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggaagtgaagagcactagttagAmino Acid Sequence: (SEQ ID NO: 75)MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGSGGGHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQND LVEYSPVTEKHLTEVKSTS

LITERATURE CITED

Reference to the following citations may assist in understanding of theinvention.

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Other Embodiments

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and scope of the invention and thefollowing claims.

All patents, patent applications, and publications referenced herein areincorporated in their entirety herein.

What is claimed is:
 1. A method for modulating a cellular activity,comprising: providing a fusion molecule to a cell, wherein the fusionmolecule is assembled by a method comprising: generating a circularpermutation of an insertion nucleic acid sequence, wherein the insertionnucleic acid sequence encodes a polypeptide that recognizes an inputsignal; and inserting the insertion sequence into an acceptor nucleicacid sequence, wherein the acceptor sequence encodes a polypeptide thatproduces an output signal provided the output signal is notfluorescence, wherein the fused insertion and acceptor sequences encodea modulatable fusion polypeptide having the output signal functionallycoupled to the input signal, wherein a change in state of at least theinsertion sequence or the acceptor sequence modulates a cellularactivity, and wherein the change in state which modulates the cellularactivity is coupled to a change in state of the respective other portionof the fusion molecule; and changing the state of the respective otherportion of the fusion molecule, thereby modulating the cellularactivity.
 2. A method for delivering a bio-effective molecule to a cell,comprising: providing a fusion molecule associated with a bio-effectivemolecule to the cell, wherein the fusion molecule is assembled by amethod comprising: generating a circular permutation of an insertionnucleic acid sequence, wherein the insertion nucleic acid sequenceencodes a polypeptide that recognizes an input signal; and inserting theinsertion sequence into an acceptor nucleic acid sequence, wherein theacceptor sequence encodes a polypeptide that produces an output signalprovided the output signal is not fluorescence, wherein the fusedinsertion and acceptor sequences encode a modulatable fusion polypeptidehaving the output signal functionally coupled to the input signal,wherein either the insertion sequence or the acceptor sequence binds toa cellular marker of a pathological condition and wherein upon bindingto the marker, the fusion molecule dissociates from the bio-effectivemolecule, thereby delivering the molecule to the cell.
 3. A method fordelivering a bio-effective molecule intracellularly, comprising:providing a fusion molecule associated with a bio-effective molecule tothe cell, wherein the fusion molecule is assembled by a methodcomprising: generating a circular permutation of an insertion nucleicacid sequence, wherein the insertion nucleic acid sequence encodes apolypeptide that recognizes an input signal; and inserting the insertionsequence into an acceptor nucleic acid sequence, wherein the acceptorsequence encodes a polypeptide that produces an output signal providedthe output signal is not fluorescence, wherein the fused insertion andacceptor sequences encode a modulatable fusion polypeptide having theoutput signal functionally coupled to the input signal, wherein eitherthe insertion sequence or acceptor sequence comprises a transportsequence for transporting the fusion molecule intracellularly, andwherein release of the bio-effective molecule from the fusion moleculeis coupled to transport of the fusion molecule intracellularly.
 4. Themethod according to claim 3, wherein either the insertion sequence orthe acceptor sequence is capable of binding to a biomolecule, andwherein binding of the fusion molecule with the biomolecule localizesthe fusion molecule comprising the bio-effective moleculeintracellularly and disassociates the bio-effective molecule from thefusion molecule.
 5. A method for modulating a molecular pathway in acell, comprising: providing a fusion molecule to the cell, wherein thefusion molecule is assembled by a method comprising: generating acircular permutation of an insertion nucleic acid sequence, wherein theinsertion nucleic acid sequence encodes a polypeptide that recognizes aninput signal; and inserting the insertion sequence into an acceptornucleic acid sequence, wherein the acceptor sequence encodes apolypeptide that produces an output signal provided the output signal isnot fluorescence, wherein the fused insertion and acceptor sequencesencode a modulatable fusion polypeptide having the output signalfunctionally coupled to the input signal, wherein the activity of theinsertion sequence and acceptor sequence are coupled, and responsive toa signal, and wherein the activity of either the insertion sequence orthe acceptor sequence modulates the activity or expression of amolecular pathway molecule in the cell; and exposing the fusion moleculeto the signal.
 6. A method for controlling the activity of a nucleicacid regulatory sequence, comprising: providing a fusion molecule,wherein the fusion molecule is assembled by a method comprising:generating a circular permutation of an insertion nucleic acid sequence,wherein the insertion nucleic acid sequence encodes a polypeptide thatrecognizes an input signal; and inserting the insertion sequence into anacceptor nucleic acid sequence, wherein the acceptor sequence encodes apolypeptide that produces an output signal provided the output signal isnot fluorescence, wherein the fused insertion and acceptor sequencesencode a modulatable fusion polypeptide having the output signalfunctionally coupled to the input signal, wherein either the insertionsequence or the acceptor sequence responds to a signal, and wherein therespective other sequence of the fusion molecule binds to the nucleicacid regulatory sequence when the signal is responded to; and exposingthe fusion molecule to the signal.
 7. The method of claim 1, wherein theinsertion sequence is inserted at a selected site in the acceptorsequence.
 8. The method of claim 1, wherein the insertion sequence isinserted at a random site in the acceptor sequence.